CA2636816C - Linear vectors, host cells and cloning methods - Google Patents

Abstract

Linear vectors derived from bacteriophage of E. coli and host cells suitable for cloning are provided. The linear vectors include a left arm comprising a left telomere and a first selectable marker, a right arm comprising a right telomere and a second selectable marker and a cloning region located between the left arm and the right arm. Optional further components of the vector include transcriptional termination sequences, multiple cloning sites and reporter stuffer regions.

Description

LINEAR VECTORS, HOST CELLS AND CLONING METHODS
INTRODUCTION
Dramatic advances in high-throughput sequencing technology have resulted in the nearly complete deciphering of the human genome and genomes of several other species.
In stark contrast to these technical achievements, there has been little improvement in the vectors or host cells used to generate the recombinant clone libraries necessary for genomic sequence analysis. Numerous genetic elements are unstable or unclonable in currently available vector/host systems due to toxicity, secondary structure, replication errors, and other poorly understood characteristics. Conventional vectors replicate to high copy number, actively induce transcription and translation of inserted fragments, and allow cloned promoters to interfere with plasmid maintenance. Moreover, conventional methods of cloning have primarily utilized supercoiled plasmid DNA, which causes instability due to torsional stress and enzymatic processing of secondary structures. Instability caused by these factors leads to sequence stacking, clone gaps, sequence gaps, and other difficulties.
The standard vectors for construction of libraries, pUC18 and its derivatives, contain many features useful for general cloning, including blue/white screening capability, large multiple cloning sites, and high copy number, as well as the ability to generate RNA
transcripts from bacteriophage promoters and single-stranded DNA from the M13 origin of replication.
However, many of these attributes are incompatible with stable maintenance of certain inserts, leading to clone gaps and seemingly "unclonablen DNA fragments. Such problematic sequences are typically characterized by high AT-content, strong secondary structure, deleterious open reading frames, or cis-acting functions (e.g., transcriptional promoters or replication origins).

Linear vectors provide an alternative to the use of circular supercoiled plasmids for cloning.
Linear vectors are not subject to the supercoiling found in circular plasmids and therefore may stably maintain inserts that have primary or secondary structures that are unstable when supercoiled. This additional stability may result in improved sequencing data and reductions in the number of sequence gaps and cloning gaps in genomic assemblies. Linear vectors also exhibit the ability to clone larger inserts using standard methods. Linear vector cloning systems may stably clone DNA in the mid-size range (10-50 kb), without the use of packaging systems required with cosmid or fosmid cloning. Linear vector cloning systems may also be used to clone fragments in the large size range (>100 kb), without the extensive vector purification needed for BAC cloning_ One linear cloning vector that has been investigated is derived from the E.
coli double-stranded DNA ("dsDNA") phage N15. In contrast to typical temperate bacteriophages that physically integrate their prophage DNA into the host's chromosome during the establishment of lysogeny, N15 replicates in the lysogen as a low-copy-number, extrachromosomal linear plasmid that has covalently closed hairpin loop telomeres. Nearly half the genome of N15, including the head and tail genes, has extensive homology with that of bacterlophage lambda (X). The elements that control transcription and determine prophage immunity have homologues in the repressor, operator, and anti-terminator of 7 and P22. The lytic development of N15 resembles that of X, resulting in virions with, 2-like morphology; and it lysogenizes at similar frequencies. The portions of phage N15 required for replication and maintenance of the linear prophage have no known equivalents in phage lambda. Conversely, the head and tail genes of phage K02 of Klebsiella oxyfoca are completely distinct from those of N15, but the genes for replication and maintenance of the linear prophages of N15 and K02 are highly homologous. (Sherwood RC et al., J.
Bacteriol., 186(6): 1818-32 (March 2004), the disclosure of which is incorporated herein by reference).
The replication of linear N15 vectors requires three components: an origin of replication (Ori), the replication initiation protein RepA, and the protelomerase TelN for resolution of the replicated telomeres. To form the prophage, the cohesive ends of the injected linear DNA
are joined to create a circular intermediate. The protelomerase recognizes a unique palindromic site (tel RL), located near the center of the previously linear molecule. It processes the linear or circularized DNA to produce a linear molecule with closed ends telL
and te1R, both in vitro and in vivo. The only N15 gene required for replication of the circular form of the plasmid is repA, which contains helicase, primase, and origin binding activities.

The origin itself is within the repA gene, and replication proceeds bidirectionally using the host E. coil DNA polymerase. N15 replication is independent of the host genes polA, dnaA, dnaJ, dnaK, grpE, and recA. The N15 genome also contains a partition system (sopBA), having homology to F' plasmid genes, but with a dispersed set of centromere sites.
The N15 virus has previously been modified into a 13.8 kb cloning vector, pG591 (SEQ ID
NO:1). The pG591 vector, which is schematically shown in FIG. 1, retains the genes = essential for replication and copy number regulation, including teIN
(protelomerase), repA
(replication factor), and cB (copy number regulator), but the phage structural genes have been removed. It also lacks the partition genes sopBA necessary for stable maintenance of the vector. Instead, the sop functions may be supplied in trans using E. coli strain DH10B31 sop, which has a chromosomally integrated N15 sop operon and anti-repressor gene, the latter under control of an arabinose-inducible promoter.
BRIEF SUMMARY OF THE INVENTION
Although it is functional as a vector, molecular cloning results using pG591 have revealed several major drawbacks. First, the left arm containing the 12 kb Notl vector fragment (teIN-repA-cB-KanR) is capable of transforming cells without the addition of an insert or the right telomere fragment, which lacks a selectable marker. Even if self-ligation of the vector is prevented via dephosphorylation, many aberrant clones and some non-recombinant clones are generated. Aberrant recombinant clones include dimers of the 12 kb fragment or circular permutations of the vector with or without various deletions between teIN and repA. Thus, because pG591 generates a high frequency of empty vector background and alternate structures, it is not acceptable for molecular cloning purposes. In addition, pG591 has only a single restriction site (Not!) available for cloning, so it is not convenient for library construction or restriction analysis of clones. Moreover, a strong promoter is directed from the right telomeric region toward the Notl site, which is likely to reduce the stability of cloned inserts by transcribing them.
The present invention relates to improved linear cloning vectors and host cells suitable for propagating the improved linear cloning vectors, kits that include both the linear cloning vectors and a strain of host cells, and methods of cloning polynucleotide sequences using the linear cloning vectors. The invention permits cloning of large or "difficult" polynucleotide sequences which may otherwise not be cloned using conventional circular plasmid vectors.
For, example, the linear vector of the invention can maintain fragments that are unstable in =

the supercolled plasmid form. The linear mode of replication imparts high fidelity replication of repeats, large palindromes, and AT-rich DNA. In addition, the invention allows for simplified molecular analysis of cloned sequences.
In one aspect, the invention provides a linear cloning vector derived from a bacteriophage capable of being maintained E. co/i. The linear cloning vector of the invention includes a left arm comprising a left telomere and a first selectable marker; a right arm cornprising a right telomere and a second selectable marker; and a cloning region located between the left arm and the right arm.
The invention also provides host cells suitable for use with the linear vector. In some embodiments, the invention provides a recombinant host cell having a polynucleotide sequence encoding a protelomerase integrated into the host cell genome.
In a further aspect, the invention provides a kit comprising a linear cloning vector of the invention and a suitable host cell.
In yet another aspect, the invention provides a method of cloning a polynucleotide sequence.
The method includes steps of processing a linear cloning vector of the invention to separate the right arm from the left arm; ligating the first end of the polynucleotide sequence to the right arm and the second end of the polynucleotide sequence to the left arm to provide a ligation product; transforming a host cell with the ligation product; and growing the transformed host cell on medium, such that selection is provided for the first and second selectable markers of the linear cloning vector.
In an additional aspect, the invention provides a method of cloning at least two distinct polynucleotides. The method includes steps of processing each of the polynucleotides to provide a linking sequence on both termini of the polynucleotides; processing a linear cloning vector of the invention to separate the right arm from the left arm and to provide a linking sequence on the terminus opposite the telomere of each arm; forming a ligation product comprising the polynucleotides and the right and left arms, wherein the arms are noncontiguous with each other and are separated by both of the polynucleotides to be cloned; transforming a host cell with the ligation product; and growing the transformed host cell on medium, such that selection is provided for the first and second selectable markers of the linear cloning vector, wherein multiplication of the host cell results in cloning of the polynucleotides.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a schematic diagram of N15 phage, plasmid pG591, and three linear cloning vectors of the invention: pNZKA, pNZKC, and pNZOC. As shown, pNZKA, pNZKC, and pNZOC carry the genes telN, repA, and cB, which are essential for replication and regulation 5 of copy number. Promoters are indicated by arrows and transcriptional terminators by "T".
Dark circles represent the telomeres. The lacZ "stuffer" fragment, situated between a pair of multiple cloning sites (MCSs), is removed before ligation to inserts.
FIG. 2 is a photograph of an electrophoretic gel used to resolve Notl digests of clones of Tetrahymena genomic DNA clones produced in pNZKA, a linear vector of the invention.
Migration of the left and right arms of the vector at 12 and 2.2 kb, respectively, is indicated.
FIG. 3 is a photograph of an electrophoretic gel used to resolve Notl digests of Oxytricha genomic clones produced in pNZKA, a linear vector of the invention.
FIG. 4 is a photograph of an electrophoretic gel used to Jesolve Noll digests of Pneumocystis genomic clones produced in pNZKA, a linear vector of the invention. FIG. 4A
shows the results of selection on kanamycin plus ampicillin and FIG. 4B shows the results of selection on ampicillin only.
FIG. 5 is a photograph of an electrophoretic gel used to resolve Notl digests of a cone snail cDNA library contained in pNZKC vectors. Set A shows 0.3 ¨ 0.7 kb inserts; set B shows 0.7 ¨ 2 kb inserts. The lane labeled "M" designates a size marker; "V"
designates empty vector control.
FIG. 6 is a photograph of an electrophoretic gel used to resolve uncut Thauera selenatis genomic DNA clones ligated in the presence of purified left arms of pNZKC.
Aberrant clones migrating as circular molecules are indicated.
FIG. 7 is a photograph of an electrophoretic gel used to resolve Notl digests of Tetrahymena genomic DNA clones produced in pNZKC and selected on kanamycin plates (upper panel) or chloramphenicol plates (lower panel). Aberrant clones migrating as circular or deleted linear molecules are indicated. Migration of left and right arms of the linear vector are indicated.
=

FIG. 8 is a photograph of an electrophoretic gel used to resolve Notl digests of Piromyces sp. E2 genornic DNA clones produced in pNZOC and selected on chloramphenicol plates.
FIG. 9 is a schematic diagram showing the process of dual insert cloning using a linear vector of the invention (pNZ-Sfi). Vector and insert fragments are not drawn to scale.
FIG. 10A is a photograph of an electrophoretic gel used to resolve restriction digests of NZSfi dual-insert recombinants. FIG 10B Is a schematic diagram showing expected restriction fragments.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following figures and examples. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The terms "including," "comprising," or "having' and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
As used in this specification and the appended claims, the singular forms "a,"
"an," and "the"
include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a vector" includes a mixture of two or more vectors. It should also be noted that the term "or" is generally employed in its sense including "and/or"
unless the content clearly dictates otherwise.
All publications, patents and patent applications referenced in this specification are indicative of the level of ordinary skill in the art to which this invention pertains It also is specifically understood that any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between. the , lowest value and' the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. The values listed above are only examples of what is specifically intended.
Linear vectors In one embodiment, the invention provides a linear cloning vector derived from a bacteriophage (also referred to herein as "phage") that is capable of replicating in E. coli as an extrachromosomal linear plasmid. Suitably, the bacteriophage used to derive the cloning vector is a temperate phage, i.e., it has a characteristic lysogenic life cycle. As used herein, a cloning vector is said to be "derived from" a bacteriophage when a genomic structure is isolated from the bacteriophage (or its host cell) and subjected to further molecular manipulation to produce a linear cloning vector. Such molecular manipulation may include deletion of structural genes or regulatory sequences, and/or introduction of one or more sequences such as promoter, operator or enhancer sequences, restriction sites, telomeres, sequences encoding selectable markers and/or transcriptional terminator sequences. As will be appreciated, a cloning vector will be considered to be "derived from" a bacteriophage whether it is directly produced (e.g., purified from a colony) or indirectly produced (e.g., through multiple rounds of cloning and subcloning, PCR amplification or synthetic methods).
Most suitably, the phage from which the linear vector is derived is suitably selected from lambda, N15, K02, PRD1 or PY54.
The linear cloning vectors of the invention include a left arm having a left telomere and a first selectable marker, a right arm having a right telomere and second selectable marker, and a cloning region located between the left arm and the right arm. As used herein, a "telomere"
refers to a polynucleotide or polypeptide structure on the end (or ends) of a linear DNA
molecule that protects the termini of the DNA from recombination and/or exonucleolytic degradation. Suitable telomeres include covalently closed ends, sequences capable of binding terminal proteins (e.g., as in PRD1), and tracts of polynucleotide repeats (e.g., poly A, C, G, T or U tracts). Examples of telomeres useful in constructing the linear vectors of the present invention include those derived from bacteriophages lambda, N15, K02, PRD1 and PY54, as well as from some linear chromosomes, e.g., those from Borrelia spp.
and Agrobacterium tumefaciens.
As used herein, a "selectable marker" refers to a phenotypic trait conferred on transformed cells that protects them from a selective agent in their environment, i.e., the growth media.
Examples of selectable markers include, but are not limited to, antibiotic resistance .markers (e.g., genes encoding resistance to kanamycin, ampicillin, chloramphenicol, gentamycin, or trimethoprim) and metabolic markers (e.g., amino acid synthesis genes or transfer RNA
genes). As is appreciated in the art, the origin of replication can also be used as a selectable marker. In some cases, the first and second selectable markers will be antibiotic resistance markers, and will be different from each other. In other cases, the first or second selectable marker may be an origin of replication (Ori). Incorporating different selectable markers on each arm of the linear vector allows for simultaneous selection of both arms among recombinant clones. Selection for both arms ensures that the structure of the recombinants is correct, having exactly one left arm and one right arm.
The cloning region of the linear vector may include a restriction site, or may be a multiple cloning site (MCS) including more than one restriction site. One or more of the restriction sites are suitably unique restriction sites, i.e., they do not occur in the vector arms. Suitably, the cloning region may include a reporter stuffer region, e.g., the lacZcc gene or a lethal gene. The reporter stuffer region may be flanked by restriction sites, or more suitably, MCSs, so that the entire reporter stuffer region may be replaced by one or more polynucleotides to be cloned. This configuration advantageously permits cloning of coding sequences which may be toxic to the cells, because strong promoters in or adjacent to the reporter region are eliminated, thus preventing transcription (and subsequent translation) of the toxic insert. In some embodiments, the total cloning capacity of the vector is approximately 50 kb.
Optionally, the linear cloning vector includes two or more transcriptional terminator regions.
As used herein and in the art, a "transcriptional terminator region," is a regulatory sequence which induces dissociation of a transcription complex in prokaryotic cells. In some embodiments, the linear cloning vector includes a pair of transcriptional terminator regions flanking the cloning region. The use of transcriptional terminator regions in this configuration reduces or eliminates transcription from the cloning region into the vector, thereby preventing interference with the function of the selectable markers, such as an antibiotic resistance coding sequence or origin of replication. Optionally, the linear cloning vector includes a third transcriptional terminator region after the selectable marker of the right arm to prevent transcription into the telomere region. Suitable transcriptional terminators are palindromic sequences which can form hairpin loop structures. The transcriptional terminator regions may be the same or different, but use of different transcriptional terminator regions may result in a more stable vector construct due to a reduced likelihood of deletions caused by recombination between identical terminator sequences.
Transcriptional terminators may be unidirectional or bidirectional. Bidirectional terminators advantageously block transcription into the insert from vector promoters and into the vector from promoters within the insert. Suitably, the transcriptional terminator following the selectable marker on the right arm Is a bidirectional transcriptional terminator. Most suitably, the transcriptional terminators are functional in the absence of host factors (i.e., are rho independent). Suitable transcriptional terminator sequences include the trpA terminator, T3 terminator, T7 terminator, rmB Ti terminator, and others as described by Reynolds, et. al, J.
Mol. Biol.
(1992) 224:31-51.
Exemplary suitable configurations for linear cloning vectors in accordance with the present invention are designated "pNZKA," (or "pJAZZ-KA," or "NZAN," SEQ ID NO:3) "pNZKC"
(SEQ ID NO:45), and "pNZOC" (or "NZTC3," or "pJAZZ-0C," SEQ ID NO: 2). These linear vector constructs are shown schematically in FIG. 1. The vectors may be provided in undigested form, or may be provided as pre-digested and dephosphorylated linear vector arms.
The linear cloning vectors described herein have at least four advantages over lambda and circular plasmid vectors. First, efficient ligation of insert to vector can be driven to completion by a molar excess of vector arms. In contrast, plasmid vectors may require numerous titrations to optimize the vector:insert ratio, as excess vector will result in independent vector molecules ligating to each end of the insert, creating a non-viable recombinant molecule. Second, in vitro lambda DNA packaging extracts limit the insert sizes to a narrow size range of approximately 35-45 kilobases (kb), whereas linear vector insertions have no minimum size, and the maximum size may be about 30-50 kb.
Third, the linear vector maintains inserts as large as those of the bacteriophage lambda vector while simplifying use and production of vectors and recombinants. Fourth, the linear vector system can be used with a simple, conventional protocol for ligation, transformation and DNA isolation, and additional components are not required (e.g., lambda packaging extracts that are required for cosmid/fosmid c)oning).
Conditions for high efficiency ligation favor the linear vector over circular plasmids.
Formation of a circular recombinant plasmid occurs in a two step reaction: an intermolecular reaction between the plasmid and insert, followed by an intramolecular reaction between the ends of the hybrid molecule to form a circle. Ligation of circular plasmid vector and large insert DNAs are typically performed in dilute reactions of about 100-150 microliters to facilitate intermolecular joining of one insert molecule to one much smaller vector molecule.
The requirement for subsequent recircularization favors smaller inserts over larger ones, requiring stringent size =selection and vector .dephosphOrYtation to .aohieve.
adbaptabie results. In contrast, ligation reactions occur most efficiently at high DNA
concentrations or under macromolecular crowding conditions. Unfortunately, these conditions favor intermolecular joining, which is optimal for forming concatamers, but not useful for creating circular plasmids. A linear vector preparation contains a left and right vector arm, each with 5 only one end capable of ligation, so high vector-to-insert ratios can be used to drive the joining reaction. Thus, linear vectors provide an improved method for generating large insert libraries by lowering the bias against large inserts.
Host cells In further embodiments, the invention provides host cells suitable for propagating the linear 10 cloning vectors. A "host cell" is any cell that may be transformed with heterologous DNA, i.e., any cell that is a competent cell. Suitably, the host cell is an E. coli cell. Suitable strains of E. coli are known, e.g., DH1OB cells or E. CLONI 10G cells (Lucigen, Middleton, WI). In some embodiments, host cells may be engineered to enhance transformation efficiency and/or maintenance of the linear vector.
Host cells may contain a coding sequence for a prokaryotic telomerase, which is referred to herein and in the art as "protelomerase" (or, alternatively, "telomere resolvase"), either on a conventional plasmid, or stably integrated into the host cell genome. A
suitable protelomerase is the N15 protelomerase, referred to herein and in the art as "TeIN."
Optionally, host cells may express protelomerase prior to transformation with the linear cloning vector. Suitably, the transformation efficiency of linear vectors in host cells expressing a protelomerase such as TeIN is 10-100 fold higher than in host cells not containing a protelomerase coding sequence.
In addition to a coding sequence for a protelomerase, host cells may further contain a coding sequence for partitioning proteins. The partitioning proteins suitably provide segregation stability to ensure accurate, non-random distribution of replicated linear plasmid molecules between the daughter cells, such that each daughter cell will receive the linear plasmid. The coding sequence for the partitioning proteins may be maintained in the host cells on a conventional plasmid, or stably integrated into the host cell genome.
Suitably, the partitioning proteins are the sopA and sopB genes encoded by the sopBA region of the N15 genome.
In addition to a protelomerase coding sequence and/or a coding sequence for partitioning proteins, host cells may further contain a coding sequence for an antirepressor. One suitable antirepressor coding sequence is the N15 antirepre.ssor gene (antA), which is known to counteract cB repression that, in tum, is believed to control the expression of RepA
protein. Thus, induction of antA leads to higher expression of RepA, thereby stimulating N15 replication and increasing prophage copy number. The N15 antA gene is suitably placed under the control of an Inducible promoter and may be contained on a plasmid or stably integrated into the genome of the host cell.
In some embodiments, the host cell contains a coding sequence for a suitable polymerase for replication of the linear vector, either contained on a plasmid or stably integrated into the genome of the host cell. As an example, the coding sequence for the PRD1 polymerase (Bamford et al., Virology 183(2):658-676 (1991)) may suitably be introduced into host cells designed to replicate linear vectors derived from PRD 1.
Kits In further embodiments, the invention provides kits containing a linear cloning vector of the invention and host cells, as described herein. Suitably, the host cells are electrocompetent or chemically competent cells modified to enhance transformation efficiency or maintenance of the linear cloning vector included in the kit. Linear cloning vectors provided in kits may be optionally pre-digested and dephosphorylated. Other optional components of the kits may include ligation buffer, ligase, control insert DNA for ligation, sequencing primers, restriction endonucleases, a phosphatase, a polymerase and/or a kinase. The kit may also suitably provide instructions for using the kit in accordance with the methods described herein.
Cloning Methods in some embodiments, the invention provides methods of cloning a polynucleotide sequence. The polynucleotide sequence to be cloned Is suitably linear, i.e., having a first end and a second end. The steps of the method include at least processing the linear cloning vector to separate the right arm from the left arm, ligating the first end of the polynucleotide sequence to the right arm and the second end of the polynucleotide sequence to the left arm to provide a ligation product, transforming a suitable host cell with the ligation product, and growing the transformed host cell on medium that selects for the first and second selectable markers of the linear cloning vector.
In some embodiments, linear cloning vectors of the invention are suitably used to clone at least two distinct polynucleotides, or insert sequences. These embodiments may be suitably employed, for example, in the cloning and expression of multi-subunit polypeptides, (e.g., the heavy and light. chains of an antibody). Such vectors are also suitably used to analyze an interaction between two or more known polypeptides (e.g., a receptor and its ligand(s)), an interaction between a known polypeptide and unknown polypeptides produced from, e.g., a library; or an interaction between two or more unknown polypeptides produced from, e.g., one or more libraries. As will be appreciated by those of skill in the art, simultaneously cloning two or more inserts also provides a means of sequencing multiple sequences via one sequencing reaction, i.e., "multiplex sequencing."
The linear cloning vectors of the invention suitably provide capacity for simultaneously cloning at least two insert sequences. In some embodiments, three inserts may be cloned.
In some embodiments, four inserts may be cloned. In some embodiments, five inserts may be cloned. In some embodiments six inserts may be cloned. In some embodiments, seven inserts may be cloned. In some embodiments, eight inserts may be cloned. In some embodiments, nine inserts may be cloned. In some embodiments, ten or more inserts may be cloned. It will be appreciated that the upper limit of the number of inserts that may be cloned using the linear cloning vectors of the invention depends on their collective size. In other words, the upper limit depends on the total capacity of the vector, e.g., 50 kb in some embodiments.
In some embodiments, at least one of the polynucleotides to be cloned is of unknown sequence. In particular embodiments, each of the polynucleotides is of unknown sequence.
In some embodiments, the sequence of at least one polynucleotide is known. In other embodiments, at least a portion of one of the sequence of at least one of the polynucleotides is known (e.g., 5, 10, 15, 20, 25 bases are known). In some embodiments, the polynucleotides to be cloned are derived from a "library," which herein refers to a collection of insert sequences derived from a source of DNA such as, e.g., an environmental source or a genome, or cDNA derived from a particular tissue or organism.
Methods of cloning at least two distinct polynucleotides, or insert sequences, include a step of processing each of the insert sequences to provide a linker sequence on both termini. A
"linker sequence," as used herein, is a sequence of nucleotides that is compatible with another linker sequence in a ligation reaction. Each of the polynucleotides to be cloned are suitably processed to provide either: a) a linker sequence on one terminus that is compatible with a linker sequence on one of the vector arms and a terminus of one other insert sequence, or b) a linker sequence on each terminus that is compatible with a linker sequence on a terminus of two other insert sequences, or c) a linker sequence on each terminus that is compatible with a linker sequence on a terminus of one other insert sequence and one vector arm. A linker sequence may be provided, e.g., by restriction, PCR

amplification and/or ligation of an oligonucleotide to the termini of the insert. The linker sequence is suitably less than 12 nucleotides in length. In some embodiments, the linker sequence is homopolymeric. Non-limiting examples of suitable linker sequences include AAA, TTT, CCC and GGG. Other examples include GTG, CAC, GTGT, and CACA. In some embodiments, one linker sequence is a blunt end. In some embodiments, the termini of each insert sequence are not compatible with each other, i.e., the insert sequences cannot self-ligate. Self-ligation may suitably be prevented by providing incompatible linker sequences on the termini or removing free 5' phosphate groups.
In a further step, the linear cloning vector of the invention is processed to separate the right and left arms. In an optional further step, the right arms may be purified away from the left arms. In a further step, the arms are treated to provide a linker sequence on the terminus opposite the telomere on each arm. Optionally, separated vector arms may be processed to prevent re-ligation, e.g., by treating the 5' ends with a phosphatase. In some embodiments, the vector arms are processed to provide "fixed orientation" multiple insert cloning, wherein the insert sequences can assemble only in a fixed orientation relative to each other and to the vector arms upon ligation.
In a further step, a ligation product is formed. The ligation product includes the insert sequences and the right and left vector arms, wherein the arms are noncontiguous with each other and are separated by the insert sequences. Most suitably, each of the insert sequences is present and present only once in the ligation product. In some embodiments, one ligation reaction provides a mixture of the desired ligation product (containing each insert and a right and left vector arm), as well as undesired ligation products lacking one or more of the inserts. However, one of skill in the art may readily determine which ligation product in the mixture is the desired product using standard techniques, for example, sequencing or restriction analysis. The single-ligation embodiment of the invention is suitable for cloning fewer polynucleotides, e.g., two or three inserts; it is also suitable for cloning a larger number of inserts, e.g., concatamers of insert sequences.
Alternatively, the ligation product may be the ultimate product of multiple ligation reactions which may be employed in a suitable scheme based on the number of insert sequences.
One suitable scheme for cloning two inserts is demonstrated in Example 12.
Suitable schemes for cloning three or more inserts are also envisioned. Some schemes may employ linkers designed such that a specific end of each insert can be ligated only to a specific end of another insert. The resulting recombinants suitably have all inserts in a fixed orientation relative, to each other and to the vector. Ligation of a . vector arm to an insert sequence suitably results in a ligation product that has only one end available for further ligation, the other end being the "inert" telomere. Thus, in some embodiments, iterative ligations are performed, wherein an additional insert sequence is iteratively added to the product of the previous ligation. A viable recombinant clone is produced only upon addition of a fragment containing the opposing vector arm. Suitable schemes for cloning additional inserts may be determined by those of ordinary skill in view of the present disclosure.
Further steps in the method of cloning at least two polynucleotides include transforming a host cell with the ligation product and growing the transformed host cell on medium, such that selection is provided for the first and second selectable markers of the linear cloning vector. It is appreciated that multiplication of the host cell results in cloning of each of the polynucleotides. Verification of the identity and orientation of the cloned polynucleotides may be accomplished by standard methods, such as, e.g., restriction analysis or sequencing.
EXAMPLES
The following examples are provided to assist in a further understanding of the invention.
The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope of the appended claims.
Example 1. Construction of host strains for linear vectors.
A standard, commercially available strain of competent E. colt (E. CLONI 10G, Lucigen, Midd(eton, WI) was used to prepare host cells for efficient transformation with linear vectors of the invention. To create one host strain (referred to herein as E. CLONI
10G-iTel), the teIN gene was PCR-amplified from phage N15 DNA (Ravin et al., J. Mol. Biol.
299(1):53-73 (May 2000), the disclosure of which is incorporated herein by reference) using the following primers:
teIN-F: GCGGATCCCGATATCCAGAGACTTAGAA (BamHI site underlined) (SEQ ID NO:
42) teIN-R: CGAAGCTTCTTTTAGCTGTAGTACGTTTC (Hind(11 site underlined) (SEQ ID NO:
43) The resulting PCR product was cloned into the BamHI/Hind111 sites of pGZ119EH, which allows cloning of the target gene under control of IPTG-inducible Ptac promoter (Lessl et al., 1992, J. Bacteriol., 174: 2493-2500, incorporated herein by reference). The recombinant vector, designated pGZ-telN, expresses teIN protein and encodes resistance to chloramphenicol. pGZ-teIN DNA was transformed into E. CLONI 10G cells by electroporation to create the chloramphenicol resistant strain E. CLONI 10G-ptel.
To integrate the teIN gene into the attB site in the chromosome of 10G cells, the fragment containing Ptac-teIN was excised from pGZ-teIN and cloned into the chromosome-5 integration vector pJW22 (Wild J, Hradecna Z, and Szybalski W (2002), Genome 12:1434-44, incorporated herein by reference), which encodes resistance to ampicillin.
The resulting integration vector, pJW-telN, was digested with Notl to excise the fragment containing Ptac-telN, which was purified by gel-electrophoresis and circularized by self-ligation. The circularized fragment was transformed into E. CLONI 10G cells (Lucigen, Middleton, WI) 10 carrying the integrase-producing plasmid pJW289t. Colonies that contained an integrated teIN gene and which had lost the pJW289t plasmid were selected as described by Wild J, Hradecna Z, and Szybalski W (2002), Genome 12:1434-44, incorporated herein by reference. The resulting ampicillin resistant strain was designated E. CLONI
10G-iTel.
The influence of teIN expression was tested by comparing the efficiency of transformation of 15 E. CLONI 10G-iTel and parental E CLONI 10G with linear pG591 DNA. The efficiency of transformation was 10- to 100-fold higher in E. CLONI 10G-iTel than in E CLONI
10G. (Data not shown).
Next, the sopBA region of N15 along with a chloramphenicol resistance marker was integrated into the chromosome of E. CLONI 100 cells to create chloramphenicol resistant strain DH10B3lsop. This strain also contains the anti-repressor AntA under control of the araBAD promoter. To add the telomerase gene to this strain, the ampicillin resistance gene of the plasmid pGZ-TeIN was replaced with a gene encoding gentamycin resistance. This plasmid was transformed into DH1OBsop31 to generate strain GTS-8 (chloramphenicol and gentamycin resistant). GTS-8 allows highly efficient transformation with pNZKA, and copy number can be induced by addition of arabinose.
A cassette containing the te/N gene, the sopBA operon, and the antA gene was integrated onto the chromosome of E. CLONI 10G cells as follows: a DNA fragment comprising phage N15 sopBA operon (under control of its own promoter) and the antA
antirepressor gene (under control of arabinose-inducib(e araPBAD promoter) was excised from plasmid pCD31sop (Mardanov A.V., and Ravin N.V., Abstracts of the conference "Lomonosov-2004", v.1, p.21, Moscow, Russia (2004), the disclosure of which is incorporated herein by reference) as an Xhol-MroNI fragment and cloned into the HindiII site of plasmid pJWtelN, described above, which contains. the teIN gene inserted into the vector pJW22, The=
-, resulting vector, pJW-telN31 sop, was partially digested with Notl to excise the fragment containing teIN-sopl3A-antA, which was purified by gel-electrophoresis and circularized by self-ligation. The circularized fragment was transformed into E. CLONI 10G
cells carrying the lambda integrase-producing plasmid pJW289t. Colonies that contained an integrated fragment comprising te/N gene, sopBA operon and antA antirepressor, and which had lost the pJW289t plasmid were selected as described by Wild J, Hradecna Z, and Szybalski W, Genome 12:1434-44 (2002), the disclosure of which is incorporated herein by reference. The resulting ampicillin resistant strain, designated E. CLONI 10G-telN31S (or, alternatively, BIGEASY TSA Cells), allow efficient transformation with the linear vector and permit induction of copy number.
Example 2. Construction of linear vectors.
a) Construction of NZCK3 A linear vector suitable for general cloning was derived from p0591 (SEQ ID
NO:1). (Ravin et al., Nucleic Acids Res. 31(22):6552-60 (2003), the disclosure of which is incorporated herein by reference) pG591 was digested with Notl and treated with a mixture of DNA repair enzymes that generates blunt, phosphorylated ends (DNATERMINTORO Kit, Lucigen, Middleton, WI). The 12 kb fragment containing the left telomere, telN, repA, and kanamycin resistance was gel isolated. pG591 was also digested with BgIII, and the 1.3 kb fragment containing the right telomere was gel isolated.
A fragment containing the lacZalpha and ampicillin genes was constructed as follows: The lacZalpha gene of the vector pEZ BAC (SEQ ID NO: 15, nucleotides 155-598) was PCR
amplified using the two overlapping forward primers T7RC-NotF (SEQ ID NO:16) and NSAS-LacZ-F (SEQ ID NO:17) plus the reverse primer NNASA-LacZ-R (SEQ ID NO:18) to create a fragment called TerZ. The ampicillin resistance gene was amplified from pSMART-HCAmp (SEQ ID NO:44, nucleotides 97-1063) by PCR with the two overlapping forward primers rrn-Fd (SEQ ID NO:19) and rrn-pCmF2 (SEQ ID NO:20) plus the reverse primer TonAmpR
(SEQ
ID NO:21). The resulting fragment was re-amplified with rrn-Fd and TonB-R (SEQ
ID NO:22) to generate the fragment TAmpT. The TerZ and TAmpT fragments were each digested with Ncof and ligated. A band corresponding to the size of the ligation product of TerZ plus TAmpT was gel isolated and re-amplified with the primers T7del (SEQ ID NO:23) and TonBR2 (SEQ ID NO:24). This PCR product was ligated into the Hincll site of pSMART
HCKan, and excised from the vector by digestion with EcoRV and BgIII. The EcoRV-BglIl fragment was ligated to the blunt 11 kb fragment and the Bgll I fragment of pG591, generating the linear vector NZAN (SEQ ID NO:3, also referred to herein as "pJA77TY-KA").. . .

The lacZ fragment of the vector NZAN was amplified by PCR with the primers LacANN-For (SEQ ID NO:25) and LacANN-Rev (SEQ ID NO:26). The resulting PCR product was re-amplified with the primers LacApSA-For (SEQ ID NO:27) and LacAsSA-Rev (SEQ ID
NO:28). The product was digested with Apal and Ascl, ligated to the 12 kb Apal fragment and the 2 kb Ascl fragment of the vector NZAN, transformed into E. CLONI 10G-pTel cells, and selected on plates containing ampicillin plus kanamycin. The resulting linear vector was designated NZASA (SEQ ID NO:4) To add additional cloning sites and binding sites for sequencing primers, the lacZ fragment was amplified from NZASA using primers LacE-SL1-F (SEQ ID NO:29) and LacA SR2-Rev (SEQ ID NO:30). The resulting PCR product was digested with Afl111, ligated to the end-repaired 10-kb Notl fragment and the 3-kb Ncol fragment of the vector NZASA, transformed into E. CLONI 10G-pTel cells, and selected on plates containing ampicillin plus kanamycin.
The resulting linear vector was designated NZAhd (SEQ ID NO:5).
To create a version of the linear vector for use with BIGEASY TSA cells, the ampicillin resistance gene of NZAhd was replaced with a chloramphenicol resistance gene.
The Ahdl restriction site in the vector backbone was also destroyed to allow cloning into Ahdl sites in the multiple cloning site. The resulting vector, designated NZCK3 (SEQ ID
NO:6), was created by ligation of four fragments. The first (left-most) fragment was the 7.8 kb Ahdl fragment of NZAhd encompassing the left telomere, te/N gene, and part of the repA gene.
The second fragment was a region of approximately 4.5 kb amplified from NZAhd by PCR
with the primers 7847-F2 (SEQ ID NO:31), which introduces a mutation that destroys the Ahdl site, and LacA-SR2-Rev (SEQ ID NO:30). This fragment was treated with Tfl DNA
polymerase in the presence of dGTP to add a single G tail to the 3' termini.
It was further digested with Spel to remove the lacZ region from the right side of the fragment. The third fragment was a region of --1.3 kb containing the lacZ region flanked by multicloning sites (MCSs), followed by the chloramphenicol resistance gene. This fragment was amplified from NZAhd by PCR with the primers LacE-SL1-F (SEQ ID NO:29) and CamTonB-Rev (SEQ
ID
NO:32); it was subsequently digested with Spel and BgIII. The fourth fragment was the 1.3 kb BglIl fragment of NZAN that contains the right telomere. A ligation reaction containing these four fragments was transformed into E. CLONI 10G-pTel cells, and recombinants containing NZCK3 were selected on plates containing chloramphenicol plus kanamycin.
. .
= . = = =
=

b) Construction of NZOC
A linear vector employing the origin of replication as selectable marker on the left arm and the chloramphenicol resistance gene as a selectable marker on the right arm was constructed. This vector, designated NZTC2 (SEQ ID NO:7), was created by ligation of three fragments. The first (left-most fragment) was a 10 kb Xbal fragment from NZASA (SEQ
ID NO:4), containing the left telomere, te/N gene, and repA gene. The Xbal restriction site was made blunt by treatment with T4 DNA polymerase in the presence of dNTPs.
The second fragment, containing the /acZ gene and flanking DNA, was amplified from NZCK3 by PCR with the primers T7-RC-Del (SEQ ID NO:8) and pCmOR (SEQ ID NO:9) and digested with Ascii. The third fragment was a -2.2 kb Ascl fragment from NZCK3 containing the chloramphenicol resistance gene and the right telomere. The ligation reaction of these fragments was transformed into E. CLON1 10G iTel cells (as prepared in Example 1) and plated on agarose containing chloramphenicol. The correct NZTC2 clone was confirmed by sequencing.
NZTC2 contained an Ahdl site in the repA gene. A derivative =lacking this site was created from three fragments. The first (left-most) fragment was the 7.8 kb Ahdl fragment of NZAhd (SEQ ID NO:5) encompassing the left telomere, te/N gene, and part of the repA
gene. The second fragment was a region of approximately 4.5 kb amplified from NZTC2 by PCR, using as forward primers a mixture of NZg7847a-F2 (SEQ ID NO:10) and NZg7847a-F3 (SEQ ID
NO:11), which introduce a mutation that destroys the Ahdl site, and the reverse primer NZ-RevB (SEQ ID NO:12). This fragment was re-amplified with NZg7847a-F2 as the forward primer and a mixture of NZ-RevA (SEQ ID NO:13) and NZ-RevC (SEQ ID NO:14) as reverse primers. This fragment was treated with Tfl DNA polymerase in the presence of dGTP to add single 3' G overhangs to the ends, and further digested with Swal to generate a blunt site on the right side of the fragment. The third fragment was the approximately 2.2 kb Swal fragment of NZTC2 that contains the right telomere. The ligation reaction of these three fragments was transformed into E. CLON1 BIGEASY TSA cells, and recombinants containing NZOC (also referred to as "NZTC3" or "pJAZZ-OC") (SEQ ID NO:2) were selected on plates containing chloramphenicol. The correct clone was confirmed by sequencing.
Example 3. Construction of a Tetrahymena thermophila 6-20 kb genomic library.
T. thermophila is a free-living, widely distributed, ciliated protozoan. The cellular, structural, and functional complexity of this organism is comparable to that of human and other metazoan cells. The macronuclear (somatic) genome consists of 160 Mb processed in vivo into -300 sub-chromosomal fragment's. Constructing libraries With =
inserts. of= >6 kb is extremely problematic for this genome, presumably because the AT content ranges from 75-85%.
The linear pNZKA vector was used to successfully clone libraries of large AT-rich fragments.
A library of 5-10 kb fragments of the Tetrahymena genome was created by ligation of sheared, end-repaired macronuclear DNA to a blunt digest of pNZKA. Of 54 clones analyzed, 51 had inserts of the expected size (data not shown).
A library was also successfully created with clones in the range of 6-20 kb, which to our knowledge, represents a library of the largest Tetrahymena fragments ever cloned.
Genomic DNA was sheared to 6-20 kb, end-repaired, gel-purified, and ligated to a Smal digest of pNZKA. Ligations were electroporated into DH10B31sop cells and selected on plates containing kanamycin plus ampicillin. The clones produced large colonies on selective plates, grew vigorously in culture, and yielded relatively high amounts of linear plasmid DNA
from standard alkaline lysis minipreps. One-fifth of the DNA from each miniprep was incubated with Notl to excise the insert and subjected to gel electrophoresis.
The results are shown in FIG. 2. Vector bands are 12 kb and 2.2 kb. Inserts are in the range of 6-20 kb.
Furthermore, sequencing reactions required only 150 ng of DNA from clones made with the linear vector.
Example 4. Oxytricha trifallax 8-20 kb genomic library.
Another genome that has been very problematic to clone is that of the ciliated protozoan Oxytricha trifallax. The DNA in the somatic macronucleus of Oxytricha is processed in vivo into "nanochromosomes" of -2-40 kb (75% AT), each fragment typically containing a single gene. Using circular vectors, previous attempts have been made to create a library of this DNA has been created for genomic sequencing, with the largest cloned insert being less than -6 kb.
To make a large-insert genomic library of the Oxytricha macronuclear genome, the DNA was end-repaired to generate blunt ends, size selected to 8-20 kb, and ligated to pNZKA. The ligation was transformed into E. coil DH10631sop cells, which contain the Sop BA region, and into E. CLONI 10G-pTel, prepared as in Example 1. The transformed cells were plated on media containing kanamycin and ampicillin, to select for both arms of the vector. (Both cell lines are resistant to chloramphenicol.) E. CLONI 10G-pTel yielded approximately 12-fold more colonies than the DH10631sop cells. For each library, 18 clones were analyzed.
As shown in FIG. 3, 15-17 clones had inserts in the range of 8-20 kb, and vector bands are = =

12 kb and 2.2 kb. A library with Oxytricha inserts of this size has not been created previously.
Example 5. Construction of a Pneumocystis carîniî 8-20 kb genomic library.
The pNZKA vector was used to clone the genomic DNA from Pneumocystis carinii, the 5 causative agent of a severe pneumonia in immuno-compromised patients. The epidemiology of P. carinii infection is poorly understood and its life cycle remains obscure. Large-scale sequencing of the P. carinii genome will help elucidate the molecular basis of the pathogenicity and speed development of drug and vaccine targets. Cloning the DNA of P.
carinii into circular vectors has previously proven problematic, even for small fragments.
10 Genomic P. carinii DNA was sheared to 8-20 kb, end-repaired to generate blunt ends, size selected to 8-20 kb, and ligated to Smal-digested pNZKA. The ligation was transformed into E. coli DH10B3lsop and into E. CLONI 10G-pTel. To show that the origin of replication can be used successfully as a selectable marker, the library was plated on media containing kanamycin plus ampicillin (FIG. 4A) and on media containing only ampicillin (FIG. 4B).
15 Colonies were randomly picked for analysis_ Each lane in FIG. 4 contains 1/5th of the DNA
from a 1.5 ml miniprep, cut with Notl to excise the insert. Vector bands are 12 kb and 2_2 kb.
Inserts were in the range of 8-20 kb. As shown in FIG. 4, both the number of colonies and the fraction of recombinants clones with the correct structure remained unchanged, regardless of the presence of kanamycin. This result indicates that the origin of replication 20 can serve as a selectable marker, as it is essential for viability of the clones. Therefore, drug selection for the left arm of the vector, which contains the origin of replication, is redundant.
Example 6. Introduction of a selectable marker on the right arm results in fewer non-recombinants.
To investigate the effects of a selectable marker on the right arm of the vector, a control insert of 2 kb, containing the /acZa gene fragment, was ligated to the vector pNZKC. Both the vector and the insert were digested with Notl, and the vector was further treated by dephosphorylation. The vector preparation was also self-ligated or unligated (i.e., incubated without ligase or insert DNA). The ligations were transformed into E. CLONI
10G-iTel cells, which are ampicillin resistant, and plated on chloramphenicol to select for the right arm of the vector_ They were also transformed into E. CLONI 10G-pTel cells, which are chloramphenicol resistant, eliminating any selection for the right arm of the vector.
. , ..=

Transformation into cells that allow selection for the right arm of the vector resulted in fewer than 0.1% non-recombinants, whereas lack of right arm selection led to nearly 20% non-recombinant, white colonies, as shown in Table 1:
Table 1. Cloning into the linear vector pNZKC with or without selection for the right vector arm.
E. cloni 10G-iTel (AmpR) E. cloni 10G-pteIN
(CamR) Pos. Control Self-ligated Unligated Pos. Control 1360 Blue 0 Blue 1 Blue 300 Blue 1 White 0 White 0 White 70 White Example 7. Construction of a cone snail cDNA library.
An example of a particularly difficult insert to clone is the cDNA derived from the poison duct of the cone snail (Conus sp.). cDNA was generated from cone snail poison duct RNA, end-repaired, and fractionated into size ranges of 0.3 ¨ 0.7 kb ("set A") and 0.7 ¨ 2 kb ("set B").
Linkers were ligated to the cDNA, and it was amplified by PCR using primers complementary to the linkers. The PCR products were ligated into pNZKC, transformed into E.
CLONI iTel cells, and plated on kanamycin plus chloramphenicol media. Plasmid DNA was isolated from randomly chosen colonies, digested with Notl, and size fractionated using gel electrophoresis.
As shown in FIG. 5, the linear vector produced only clones in the expected size range of 0.3 to 2 kb. Ligation and transformation of the cone snail cDNAs into conventional circular plasmicls resulted in predominantly empty vectors or inserts of <100 bp (data not shown).
Example 8. Linear vectors containing only the left arm convert to a circular plasmid.
pNZKC was digested with Smal, and the 12 kb left arm was gel purified away from the /acZ
stuffer region and the right arm. 1-2 kb fragments of DNA isolated from the genome of Thauera selenatis were prepared by shearing (using a HYDROSHEAR device, Gene Machines, San Carlos, CA). The fragments were end-repaired, gel-purified, and ligated to a Smal digest of the purified left arm of pNZKC. Ligations were electroporated into E. CLON1 10G-iTel cells and selected on plates containing kanamycin. Eighteen kanamycin resistant colonies were randomly picked for analysis and 1/5th of the DNA from a 1.5 ml miniprep was resolved using agarose gel electrophoresis.
=
=

The results, shown in FIG. 6, show that at least 10 out of the 18 clones were converted to a circular plasmid, showing supercoiled and relaxed circular forms, while the remaining 8 clones appeared to be linear. In all 18 cases, the clones were not able to survive on plates containing chloramphenicol, indicating they lacked a right arm. Aberrant clones are indicated by "*" in FIG. 6.
Example 9. Selection for both left and right arms favors the linear vector form.
Tetrahymena genomic DNA was sheared to 4-10 kb, end-repaired, gel-purified, and ligated to the left and right arm of a Smal digest of pNZKC. Ligations were electroporated into E.
CLONI 10G-iTel cells and selected on plates containing kanamycin only (which selects only for the left arm of the vector) or kanamycin plus chloramphenicol (which selects for both arms of the vector). Colonies were randomly picked for analysis, and 1/5 of the DNA from a 1.5 ml miniprep was cut with Notl to excise the insert and resolved using agarose gel electrophoresis.
As shown in FIG. 7, when the right arm was not selected by chloramphenicol, 8 out of 18 clones appeared to be linear molecules missing the expected 2.2 kb Notl fragment, which represents the right arm of the vector (FIG. 7, upper panel), and 2 of the clones (lanes 6 and 10) appeared to be circular plasmids instead of linear vector. In contrast, when the right arm was under selection by chloramphenicol, 17 out of 18 clones contained the expected 2.2 kb Notl right arm fragment (FIG. 7, lower panel). Since the origin of replication is essential for viability of the vector, the left arm is under selection regardless of the antibiotic used.
Example 10. Improved transformation efficiency with strains containing the teIN
gene.
Genomic DNA from Oxytricha trifallax and from Pneumocystis carinii was sheared to 8-20 kb, end-repaired, gel fractionated, and purified. The linear vector pNZKA was digested with Smal and dephosphorylated. Approximately 300 ng of each prepared genomic DNA
was ligated in separate reactions to 50 nanograms of the prepared linear vector.
The ligation reactions were heat-inactivated and transformed into host strains that had been rendered electrocompetent. The host strains included E. CLONI 10G-pTel, which contains a te/N
protelomerase coding sequence, and DH10B31 sop cells, which does not contain a te/N
coding sequence. One-tenth of the transformed cells were plated onto media containing kanamycin, ampicillin, XGAL, and IPTG. After overnight growth, each ligation reaction yielded ¨12-fold more colonies in the teIN strain (Table 2).
õ .
.. =

Table 2. Improved transformation efficiency of the linear vector in a host strain containing telN.
pNZKA plus Pneumocystis DNA pNZKA plus Oxytricha DNA
E. CLONI 10G-pteIN 800 2400 DH 1 OB31sop 62 200 In separate experiments, the two host strains were shown to have similar transformation efficiency when electroporated with pUC19 DNA, indicating the ability to take up DNA was similar for the two strains. The linear vector was maintained in both strains of cells after several rounds of freezing, dilution, and re-growth, indicating that the linear plasmid was stably maintained in both strains.
Example 11. Construction and use of a single-antibiotic-resistant linear vector Piromyces sp.E2 is a fungus of the phylum Chytridiomycota. The genomic DNA
from this microbe is approximately 85% AT, and cloning fragments even as small as 2 kb is very difficult in standard circular vectors. In contrast, fragments of this genome as large as 2-6 kb could be successfully cloned in the NZOC vector.
Ten micrograms of Piromyces DNA was physically sheared to 2-6 kb using a HydroShear Device (Gene Machines), and the ends were repaired using the DNATERMINATORO
kit (Lucigen, Middleton, WI). The DNA was quantitated against a DNA mass standard using gel densitometry software (Alpha Innotech, San Leandro, CA), and ligated to a blunt digest of the pJAZZO OC vector. The ligation reaction contained approximately 100 ng of insert DNA, 50 ng of digested pJAZZO OC, ligase buffer, and 2 U T4 DNA ligase. The reaction was incubated at room temperature for 2 hours, heat treated for 15 minutes at 70 C, and used to transform electrocompetent E. CLONIO BIGEASYTM TSA cells. Cells were spread on to an agar plate containing 12.5 ug/ml chloramphenicol, XGAL, and IPTG. Linear plasmid DNA
was isolated using standard alkaline lysis purification with binding to diatomaceous earth.
The DNA was digested with Not I and assayed by agarose gel electrophoresis. As shown in FIG. 8, nearly all of the recombinant clones contained inserts of 2-6 kb.
Twelve samples were sequenced to confirm that they contained genomic DNA from Piromyces. The AT
content of some of these clones approached 96%; obtaining clones with this level of AT
content has not been reported previously.
= =

Example 12. Dual-Insert Cloning in a Linear Vector The present Example describes construction of a dual-insert library in a linear vector. The insert DNAs were defined fragments of 10 kb amplified from E. coli genornic DNA by PCR
using the Phusion polymerase (New England Biolabs) according to the manufacturer's recommendations. The primers used for PCR amplification were:
Primer 1: TTCTTATGGCCAGGGAGGCCGCTCTGGGTATAAGCGTAAGG (SEQ ID NO:33) Primer 2: AACTAGTGGCCAGGGAGGCCATCAGCCAGGCGACGAATCAG SEQ ID NO:34) Primer 3: GGACTTGGGCCACCCAGGCCTTGTAAATGCAGTATGGATTG (SEQ ID NO:35) Primer 4: ATCCTAGGGCCACCCAGGCCAGATATTGGAGAGTTGCACCAG (SEQ ID
NO:36) One PCR product, termed "EC39" was amplified using Primers 1 and 2; a second product, "EC40," was amplified using Primers 3 and 4. The primers also contain the recognition site for the restriction enzyme Sfil (underlined above), which after digestion leaves a 3 base pair overhang on the 3' strand of the double-stranded DNA amplification product.
EC39 was digested with Sfil to produce a 3' extension of ¨CCC; digestion of EC40 by Sfil created a 3' extension of ¨GGG. The digested products of the EC39 insert are therefore not able to ligate to themselves to form concatamers. Similarly, the digested EC40 products cannot self-ligate. Consistent with the scarcity of Sfil sites in most genomes, the regions chosen for amplification do not have internal Sfil sites. The 10 kb Sfil digestion products were purified and quantitated.
Vector pNZ-Sfi (SEQ ID NO:37) was derived from pNZKA by replacing the multiple cloning sites and the lacZ stuffer of pNZKA with a DNA fragment containing the lacZ
stuffer flanked by different multiple cloning sites, including sites for the restriction enzyme Sfil. The new lacZ stuffer was generated by PCR amplification of the lacZ region of the vector NZAhd using the primers lacFSfi (SEQ ID NO:38) and lacRSfi (SEQ ID NO:39). The primers were phosphorylated with T4 polynucleotide kinase prior to performing the PCR. The resulting PCR product was purified and ligated to a Sma I digest of the vector NZAhd.
The ligation reaction was transformed into E. cloni GTS-8 cells, and transformants were selected on agarose plates containing ampicillin, kanamycin, XGAL, and IPTG. The correct pNZ-Sfi clone was confirmed by sequencing.
= = - = ===

The Sfil sites of the vector were designed such that digestion with Sfil creates a 10-kb left arm with a 3' extension of ¨GGG, a 2-kb right arm with a 3' extension of ¨CCC, and a 0.5 kb lacZ stuffer fragment. The ¨GGG extension on the left arm is compatible with the ¨CCC
extension created by Sfil digestion of EC39; similarly, the right arm is compatible with the Sfil 5 digest of EC40. The 5' phosphates were removed from the vector Sfil fragments by treatment with Calf Intestinal Phosphatase to prevent re-ligation of the vector arms. The digested vector fragments were fractionated on an agarose gel, and the bands were individually excised, purified, and quantitated.
Dual insert cloning was performed as diagrammed in FIG. 9. The Sfil-digested left arm was 10 ligated to an equimolar amount of Sfil-digested EC39. In a separate ligation reaction, the Sfil-digested right arm was ligated to an equimolar amount of Sfil-digested EC40. After allowing the ligation reactions to proceed to at least 50% completion, aliquots of the two ligation reactions were combined with each other. Further incubation was carried out to facilitate ligation of the left arm/EC39 molecules to the EC40/right arm molecules. The final 15 ligation reaction was heat-inactivated, and the products were transformed into GTS 8 cells (Lucigen, Middleton, WI). An additional ligation reaction was performed with only the Sfi-l-digested left and right vector arms to measure the frequency of self-ligation.
One-tenth of the transformants were plated on media containing kanamycin and ampicillin to select for both arms of the linear vector. The plates also contained XGAL plus IPTG to 20 screen against uncut vector or recombinants containing the lacZ stuffer fragment. The dual-insert ligation/transformation reaction produced ¨2200 white colonies and 26 blue colonies.
The self- ligation/transformation yielded 208 white colonies and 23 blue colonies.
Thirty white colonies were randomly selected from the dual-insert plate, and grown overnight in TB media containing kanamycin plus arabinose. Plasmid DNA was isolated by standard 25 alkaline lysis methods, and restriction-digested with Spel, NgoMIV, or Noll Spel has a single recognition site near the cloning site of the left arm and one site within the EC40 fragment.
NgoMIV has a single site within the EC39 fragment, but no sites in the vector or in EC40.
Notl has a site near each of the cloning sites, and no sites within EC39 or EC40; it therefore excises the entire dual insert. In all thirty clones analyzed, restriction analysis with these enzymes confirmed the presence of exactly one copy of each fragment and each vector arm in the expected relative positions of Left arm-EC39-EC40-Right arm, as shown in FIG. 10.
=

Example 13. Derivation of a linear vector from phage PRD1.
Genomic DNA from phage PRD1 is digested with BsrBI to remove the left telomere and its associated terminal protein from the genomic DNA. The 3-kb BsrB1 fragment is isolated by agarose gel electrophoresis. Another aliquot of phage genomic DNA is digested with Xmnl to remove the right telomere and its associated protein. The 1-kb Xmnl fragment is isolated by agarose gel electrophoresis. PCR is used to amplify a DNA fragment containing a selectable marker and, optionally, a visual screening marker. Creation of such a fragment, containing the TAmpT and TerZ segments, is described in Example 2.
The DNA polymerase of phage PRD1 (GenBank ACCESSION NC 001421) is amplified by PCR with the primers PRD1 POL- F (SEQ ID NO:40) and PRD POL R (SEQ ID NO:41).
The 1.7 kb PCR product is purified, digested with Sphl, and cloned into a bacterial expression vector (e.g., pET24, Novagen).
The PRD POL expression vector is transformed into E. CLON1 cells, and a clone expressing the PRD1 polymerase gene is confirmed by sequence analysis. Expression of the polymerase is verified by presence of an additional band at approximately 65 kD on an acrylamide gel.
Alternatively, the PRD1 polymerase gene is appended to a promoter sequence and integrated into the genome of E. CLONI cells, using e.g. methods described for integration of the teIN gene in Example 1.
The PRD1 expression clone is made competent by standard techniques. A ligation reaction containing the 1-kb BsrB1 fragment, the TAmpT-TerZ fragment, and the 3-kb Xmnl fragment is transformed into the competent PRD1 expression cells. Colonies that express blue color and are ampicillin resistant are selected for further growth. The presence of the PRD-AmpLacZ vector is confirmed by restriction analysis and sequencing of plasmid DNA.

, SEQUENCE LISTING
<110> LUCIGEN CORPORATION
<120> LINEAR VECTORS, HOST CELLS AND CLONING METHODS
<130> 33089-2003 <140> CA2,636,816 <141> 2007-01-12 <150> US 60/758,479 <151> 2006-01-12 <150> US 60/747,733 <151> 2006-05-19 <160> 45 <170> PatentIn version 3.3 <210> 1 <211> 13165 <212> DNA
<213> Artificial <220>
<223> pG591 <220>
<221> misc_feature <222> (10741)..(10741) <223> n is a, c, g, or t <220>
<221> misc_feature <222> (10987)..(10987) <223> n is a, c, g, or t <400> 1 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 , tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 ggcgagaacg ggcagtggca gctgaagata gagacacctg caatcgtcct gcctgatgaa 1800 gaatccgttg agaccatcga cgaaccggat gatgagtccc aagacgacga gctggatgaa 1860 gatgaaattg agctcgacga gggtggcggc gatgaaccaa ccgaagagga agggccagaa 1920 gaacatcagc caactgctct aaaacccgtc ttcaagcctg caaaaaataa cggggacgga 1980 acgtacaaga tagagtttga atacgatgga aagcattatg cctggtccgg ccccgccgat 2040 agccctatgg ccgcaatgcg atccgcatgg gaaacgtact acagctaaaa gaaaagccac 2100 cggtgttaat cggtggcttt tttattgagg cctgtcccta cccatcccct gcaagggacg 2160 gaaggattag gcggaaactg cagctgcaac tacggacatc gccgtcccga ctgcagggac 2220 ttccccgcgt aaagcggggc ttaaattcgg gctggccaac cctatttttc tgcaatcgct 2280 ggcgatgtta gtttcgtgga tagcgtttcc agcttttcaa tggccagctc aaaatgtgct 2340 ggcagcacct tctccagttc cgtatcaata tcggtgatcg gcagctctcc acaagacata 2400 ctccggcgac cgccacgaac tacatcgcgc agcagctccc gttcgtagac acgcatgttg 2460 cccagagccg tttctgcagc cgttaatatc cggcgcagct cggcgatgat tgccgggaga 2520 tcatccacgg ttattgggtt cggtgatggg ttcctgcagg cgcggcggag agccatccag 2580 acgccgctaa cccatgcgtt acggtactga aaactttgtg ctatgtcgtt tatcaggccc 2640 cgaagttctt ctttctgccg ccagtccagt ggttcaccgg cgttcttagg ctcaggctcg 2700 acaaaagcat actcgccgtt tttccggata gctggcagaa cctcgttcgt cacccacttg 2760 cggaaccgcc aggctgtcgt cccctgtttc accgcgtcgc ggcagcggag gattatggtg 2820 tagagaccag attccgatac cacatttact tccctggcca tccgatcaag tttttgtgcc 2880 tcggttaaac cgagggtcaa tttttcatca tgatccagct tacgcaatgc atcagaaggg 2940 ttggctatat tcaatgcagc acagatatcc agcgccacaa accacgggtc accaccgaca 3000 agaaccaccc gtatagggtg gctttcctga aatgaaaaga cggagagagc cttcattgcg 3060 cctccccgga tttcagctgc tcagaaaggg acagggagca gccgcgagct tcctgcgtga 3120 gttcgcgcgc gacctgcaga agttccgcag cttcctgcaa atacagcgtg gcctcataac 3180 tggagatagt gcggtgagca gagcccacaa gcgcttcaac ctgcagcagg cgttcctcaa 3240 tcgtctccag caggccctgg gcgtttaact gaatctggtt catgcgatca cctcgctgac 3300 cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 , taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 , ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gagtcgacct gcaggcatgc aagcttcctg aatcgcccca tcatccagcc 10260 agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 10320 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 10380 tcaactcagc aaaagttcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat 10440 gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa 10500 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 10560 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 10620 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 10680 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 10740 nttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 10800 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 10860 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 10920 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 10980 tttcccnggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 11040 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 11100 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 11160 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 11220 atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg tttcccgttg 11280 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 11340 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct 11400 ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 11460 cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 11520 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggaagc 11580 ttgcatgcct gcaggtcgac tctagaggat ccccgagaac ccgataatcg ctaccagtga 11640 tgatggctgt tttgcggcgg cgtgagccat cggcaatttc gataatgcct gacgtccttc 11700 tggcgaacgc ggggttctgc tgtcctgaag tgaggaatga agggataagg tcggccagcg 11760 ctgattcgtt cagcaattcc tgatcacgtt cattaccgag ccaaaccatt gtggcctttt 11820 cgactttatc agcaggaatg gtttccagct taaaagtcac gttgcggccg caaaccggta 11880 gcgtaatgct attcccggac aacgaactta atttgctctg taagtcgcca accatcggcg 11940 aaaccgatgg cgctttttgc ggcgcattat ggttattgct catgaacgtt tcgatgttgg 12000 gcgcattttt taatatagag cgattcttca tagttagtcc tcccaacgag gtttgattag 12060 atctgtttca atgcggtgaa gggccaggca gctggggatt atgtcgagac ccggccagca 12120 tgttggtttt atcgcatatt cagcgttgtc gcgtttaccc aggtaaaatg gaagcagtgt 12180 atcgtctgcg tgaatgtgca aatcaggaac gtaaccgtgg tacatagatg cagtcccttg 12240 cgggtcgttc ccttcaacga gtaggacgcg gtgcccttgc aaggctaacc attgcgcctg 12300 gtgtactgca gatgaggttt tataaacccc tcccttgtgt gacataacgg aaagtacaac 12360 cgggttttta tcgtcaggtc tttggtttgg gttaccaaac acactccgca tatggctaat 12420 ttggtcaatt gtgtagccag cgcgacgttc tactcggccc ctcatctcaa aatcaggagc 12480 cggtagacga ccagcttttt ccgcgtctct gatagcctgc ggtgttacgc cgatcaggtc 12540 tgcaacttct gttatacccc agcggcgagt aatacgacgc gcttccgggc tgtcatcgcc 12600 gaactgtgcg atggcaatag cgcgcgtcat ttcctgaccg cgattgatac agtctttcag 12660 caaattaatt aacgacatcc tgtttcctct caaacatgcc cttatctttg tgtttttcat 12720 catactttac gtttttaaag caaagcaaca taaaaaaagc aaagtgactt agaaaacgca 12780 aagttaaggt tcaaatcaat tttttgatgc gctacagaag ctatttagct tcatctaagc 12840 gcaacggtat tacttacgtt ggtatattta aaacctaact taatgatttt aaatgataat 12900 aaatcatacc aattgctatc aaaagttaag cgaacatgct gattttcacg ctgtttatac 12960 actttgaggc atctctatct cttccgtctc tatattgaaa cacaatcaaa gaacatcaat 13020 ccatgtgaca tcccccacta tctaagaaca ccataacaga acacaacata ggaatgcaac 13080 attaatgtat caataattcg gaacatatgc actatatcat atctcaatta cggaacatat 13140 cagcacacaa ttgcccatta tacgc 13165 <210> 2 <211> 12827 <212> DNA
<213> Artificial <220>
<223> NZOC (NZTC3 or pJAZZ-0C) <400> 2 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 OI 35rd ozvE 3e6oe65364 Boo4.6q41.4q quE.E.BqpqvB 6456o66pp 66.456qepo5 qE5qp65oqo 09EE Eolo;qa6e6 Dp6o68qppt, 6DBErqD663P vppoe65pEo PP6PopErqp6 6E.DyTeE66o HEE ov6;o6pqop vogpEobqso qq6Bqolpp6 qoppqqqE,DE. 65qoppE.6po Epooqpq63q otzE upoqopqq6D E.E.po6vobqo oppoqqa6p6 pvaeopo6.26 rp6p6.466o6 gEceqpElpbbq 08TE DE-eqroqDo6 6q6o6PoPqs vpoEcgooqqo 6po600qq6E, p5e.p6qopP6 pEoBoSoqq6 Om P.64606.1DDQ qa6p6a6po6 36.e.E6Eceop 666-eps6sol 363oaeolql pHoppoqop 090E 6354qpoqo 3bvEp6p663 PEPPPP&;PP p6;p3qqqa6 6q666Eq-246 DOOPOOPP6P
000E PovEopropp o;666propy vuopopeoae opqr-TeEreop oBvpbqvuog qvgvq356qq 0t6z 666.ep5poTe pEcTepobopq qa6pooTe61. poTeolqqq1 ppoq666.26o 3pvt.qq6631.
088z 336.4641q3g EpPoqp6poq uppE,6qopoq qopqqgpoyD p-eqvEopqqp BypopEcebuq OZ8Z 51661-eq;p6 6p6636up6E. oboq636opy 3qqq6q3po3 gEoq64o66y 33633peE63 091,z 64q3po3opo q63q1.63qop vpBuDE)6qa6 pq-2.66opqqq qq6opEoqoP qP06PPUPOP
ooLz 631386-e333 66e143q463 6633e33q56 qEcepoqBepo Boobqoqqqo qqoqqbppEo OV9z 3oo.6.6PoTeq qq63q6-Teqo 6z6qqqappv pEcgovq86ou qq6D5Te3oo ppqoBoo6oe ossz Bsoogpop6p Bv66366363 66-epEcqo3qq 66 6E
qq6E6qqpqg BEopopTepq ozsz P6e66B3o61 Te6qp6DE6D go5PDBoE6D 3qpqp-eq353 3636 )3 6336-eb2333 09T7z Eqq8TeD6DP ov6eq6pqq5 opoqoEpoSE 08360qP0Pq 3PP6OPOD60 osbobBooqo 0017z ulPopEceupp 33g3l36u36 6D1-26q66pq PqueD4E-453 3ggSvpoqD4 qooPpErep66 ot,Ez -;36.461PPPP olo6uop65q spoqqqqoSE. 331.14606.e-4 p66q6pqq.46 Pq4.6quEo66 08Zz q35oqppo64 oqq444Pqop oppoDa6qp.6 Hoqqvvvqg 3.6.65.6DET-ep q6o6oppogq ozzz op66Be36qo pEopogEop6 pqpopEeoyq Dero6qp6p3 Egovpy6.636 6pqq-e66pE6 09TE 60P6S6PP36 qpooDTepoo vqpooq6qop 66p6.4.4t,qqq. morqp66.466D Teeqq6-ootz DPOOSPPPUE. E.PPUgDE.PDP qppq6DevE) 681.PpEopqp .636TePD6DD 66.4E-43336u otoz Te6006op3o 6603.466qop EcTeqq-epBuv p6E,TeEopqu p6qqq6p6Pq pBypougEoP
0861 PE6o.e66663 PP4PPPPPPO EqopBsepqq. D.T6opaesev qpi.obqoppo 36poleoPPE1 0z61 PE.5po3666p EB5P6PP600 PP33EP64P6 3E63664666 .2.63.e6pqpEce 6qqvu-e6qP6 0981 p.e5Te5.6.335 vEop6DPEcep opoq6p6Te6 TeBBooseED p63.4roppEce.
6q45ooqp.e6 0081 re5ge6q335 googbogPeo BloppDBE,E. pgu5es6qp6 Po66.46p366 5oyy6pE366 oT7LT qq63qq5e35 666qq.236Te 63.6po6qq.q. 6p66qoppor6 Boo6pqqp6.4 uEopBooDET.
0891 14Tes...eqqqo 366633l333 E36P3PP3OP PqPUPPPOBP 3Tep33p56.2 a6p66q.66qo onT aeoEcep;q6o opp.e6qpopq DT633.35a6E, q36DP.646Ece Bupobqqqa6 6eop6Tepp6 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 , ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gacagtccag ttacgctgga gtcactagtg cggccgcgac aacttgtcta 10260 gggcccaatg gcccgggagg cctacttaag taagccggct tagctagcgg gacaggtttc 10320 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 10380 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggac 10440 aacaatttca cacaggaaac agctatgacc atgattacgc caagctattt aggtgagact 10500 atagaatact caagcttgca tgcgatacgt atcgttaacg atggatccga cgcacgtgcg 10560 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac 10620 tgggaaaacc ctggcgtcac ccaacttaat cgccttgcag cacatccccc tttcgccagc 10680 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagctgaatg 10740 , gcgaatctta agtaggcctc ccgggccatt agacttgaag tcaagcggcc gctacaactg 10800 gaccttgctg gtacatagaa ctgattaact gaccatttaa atcataccaa catggtcaaa 10860 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt taatctgatc ggcacgtaag 10920 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc 10980 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt 11040 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg 11100 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa 11160 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc 11220 ggaatttcgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg 11280 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga 11340 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct 11400 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt 11460 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt 11520 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt 11580 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta 11640 ctgcgatgag tggcagggcg gggcgtaacc taggtgacag aagtcaaaag cctccggtcg 11700 gaggcttttg actttctgct agatctgttt caatgcggtg aagggccagg cagctgggga 11760 ttatgtcgag acccggccag catgttggtt ttatcgcata ttcagcgttg tcgcgtttac 11820 ccaggtaaaa tggaagcagt gtatcgtctg cgtgaatgtg caaatcagga acgtaaccgt 11880 ggtacataga tgcagtccct tgcgggtcgt tcccttcaac gagtaggacg cggtgccctt 11940 gcaaggctaa ccattgcgcc tggtgtactg cagatgaggt tttataaacc cctcccttgt 12000 gtgacataac ggaaagtaca accgggtttt tatcgtcagg tctttggttt gggttaccaa 12060 acacactccg catatggcta atttggtcaa ttgtgtagcc agcgcgacgt tctactcggc 12120 ccctcatctc aaaatcagga gccggtagac gaccagcttt ttccgcgtct ctgatagcct 12180 gcggtgttac gccgatcagg tctgcaactt ctgttatacc ccagcggcga gtaatacgac 12240 gcgcttccgg gctgtcatcg ccgaactgtg cgatggcaat agcgcgcgtc atttcctgac 12300 cgcgattgat acagtctttc agcaaattaa ttaacgacat cctgtttcct ctcaaacatg 12360 cccttatctt tgtgtttttc atcatacttt acgtttttaa agcaaagcaa cataaaaaaa 12420 gcaaagtgac ttagaaaacg caaagttaag gttcaaatca attttttgat gcgctacaga 12480 agctatttag cttcatctaa gcgcaacggt attacttacg ttggtatatt taaaacctaa 12540 cttaatgatt ttaaatgata ataaatcata ccaattgcta tcaaaagtta agcgaacatg 12600 ctgattttca cgctgtttat acactttgag gcatctctat ctcttccgtc tctatattga 12660 aacacaatca aagaacatca atccatgtga catcccccac tatctaagaa caccataaca 12720 gaacacaaca taggaatgca acattaatgt atcaataatt cggaacatat gcactatatc 12780 atatctcaat tacggaacat atcagcacac aattgcccat tatacgc 12827 <210> 3 <211> 14600 <212> DNA
<213> Artificial <220>
<223> NZAN (pJAZZ-KA or pNZKA) <400> 3 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 L132Vd 090E 636qTepTqo D6P6P6P5.60 p5ePPE61PP pEcqopqqq36 64655pTE-45 DOOPOOPP6P
000E POPEDDPDOP D.1656oppop ppopopEoEre 3p4P1P6e3p 36p364.epoq qP1E-4366T4 ot6z 6.6.6pP6p3Te 36qppoEopq qa6pooqpbq poqpaqqqq1 pPoq66E,P63 oppq-4663q 088g Do6q6qq41q EppoTeEopq pooBbloopq qouqqqpoyo Dpqp5poqq. 666P1 ciez 6q664pqqp6 E.E.6636yDEE, DboqEoboop oqqq6lopop q63q6.4o66E. 33E33py663 09Lz Elqoppopeo q6pqq5pqop puBvp56436 pq-265poggq .416poboqov 1P06PPPPOP
OOLZ 6ogo68polo E.E.pqp16D 6666 q6pooqbeop 633613iqq3 11o116sP63 0V9Z D0066voqP1 qq6pq6qpqo 5q6qqapvpp pEclot-ILEop 4q6o6qvpoo Pv1363363e 08sz 6pooqv336E. 6e66365363 E5eo6gooqq. 6654PEq.533 qq.65511eql 55oeooquoq ozsz e6e6663364 qu6qP6o663 q3Ece353563 ogegypqq53 3Ecep513441 6006u5epoo 0917z 541.64.eo53v pa6pq.63qq.6 popqa6poEu a6a6oquosq oevbopoo.63 ae63.6633qo ootz yqropbrupp poqoloBuo.6 6oTe.6.366pq pgupoTegED oqqbpooqoq qopPo6vD65 ot,Ez 4364.6qupPy 3go6p3oB6-4 ppoqqqqoEu opm4.46paeq pB.6.163qqq.E. e6e566 ogEz Qp6oTepo64 oqq1T3pqop oppooBbqp6 6.6pqqpr-eqg p6666p5epv q6o6poopql oz zE ov668ppEqo pEopoq6=6 DTeopE6opq oppo6goEmo 6qouvs6635 6-eq1p56E.P6 091z 63p666pp36 q0000Tepop pqoppq6qop 66-e6qqpqqq qqq366166o Tepqq6;653 00-E oE'opEcePPPE, UPEPQDBPDP lop45opup5 6Ee 63E6op 66qpqoppae (÷oz -1P6oDE3oo0 66336633 5pqqp36.ep p6Eqs5opqp p6qqq6p6E.q P6E.PoygEoy 0861 P66DP666.60 PPTPPPPPP3 EqaoSppoqq. ol8poop-Eep qoqp6qop-ep DEceDqE.0-E,P6 0z61 PPEPD0666e P66P6PP500 PPOOPPEc4P6 DB63E6.4666 -e6oPEoqp6e 5qqpr.E6.4E.E.
0981 pvEcTe66.4o6 p6opEoe6PP popq6-26qp.6 qp66pDPE,Eo E.53Te33u6v .6.44.6poquu5 009T Pp61p6qoa6 looqboTepo 5qoppou6e6 vq-26.epEgo6 po6.6q6po66 6osp6v6o66 oi7LT qqEoqq6u3.6 66.6qqPo.6.48 5qp.6335.4qq 6.e.6.6qp3-eq5 5oa6pqq-e6q peoP6oDa6p 0891 qqqp-epqqqo 35.663DqDqD POSPOPPOOP PTePPPPDEP oTepoop.66p oEce.65-45.6qo ozgi Epobvpqq6o oPrp6q.sooq D.4.63D-1.6o5.6 qa6op5q66P 6eop.61-qqD6 5poo6TePP6 0961 qp5op6.6qop pe6po6qoqo 56q6Eqo5BE, DDEOPPPP61 p5566.4q6ps, 6q3pP6366.4 0061 DOPP6PD0q0 qqpreDo661 35pp3qq6pD 6PP1Pqae0.6 qa6POODPDP P6P6qP6DP6 ottT Dppe6SoqD1 1p6p6blrog qpq46161p6 6pEop5oq6o pppPRBE,q65 3p333,p6316 08ET o600q4Dqq5 Te6p6Teqqo 6olpo53q36 opqqqpqp5o 63o6elp5ev -2.4.eqq.46T6o ozET 1633-e6q.E.53 6.631qqlqu3 Teupqq665q qopoppgqqp 35PPRP36P4 lqq-eqp6Tep cctccccgga tttcagctgc tcagaaaggg acagggagca gccgcgagct tcctgcgtga 3120 gttcgcgcgc gacctgcaga agttccgcag cttcctgcaa atacagcgtg gcctcataac 3180 tggagatagt gcggtgagca gagcccacaa gcgcttcaac ctgcagcagg cgttcctcaa 3240 tcgtctccag caggccctgg gcgtttaact gaatctggtt catgcgatca cctcgctgac 3300 cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 i ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 , tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gagtcgacct gcaggcatgc aagcttcctg aatcgcccca tcatccagcc 10260 agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 10320 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 10380 tcaactcagc aaaagttcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat 10440 gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa 10500 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 10560 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 10620 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 10680 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 10740 cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 10800 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 10860 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 10920 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 10980 tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 11040 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 11100 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 11160 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 11220 atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg tttcccgttg 11280 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 11340 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct 11400 ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 11460 cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 11520 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggaagc 11580 ttgcatgcct gcaggtcgac tctagaggat ccccgagaac ccgataatcg ctaccagtga 11640 tgatggctgt tttgcggcgg cgtgagccat cggcaatttc gataatgcct gacgtccttc 11700 tggcgaacgc ggggttctgc tgtcctgaag tgaggaatga agggataagg tcggccagcg 11760 ctgattcgtt cagcaattcc tgatcacgtt cattaccgag ccaaaccatt gtggcctttt 11820 cgactttatc agcaggaatg gtttccagct taaaagtcac gttgcggcca tcaagcttga 11880 attcgtacgc agaaaggccc acccgaaggt gagccagtgt gattacattt gcggccgcat 11940 ttaaatgggc ccgggacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 12000 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 12060 tgttgtgtgg aattgtgagc ggacaacaat ttcacacagg aaacagctat gaccatgatt 12120 acgccaagct atttaggtga gactatagaa tactcaagct tgcatgcgat acgtatcgtt 12180 tacgatggat ccgacgcacg tgcgaattcg ccctatagtg agtcgtatta caattcactg 12240 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg tcacccaact taatcgcctt 12300 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 12360 tcccaacagt tgcgcagctg aatggcgaat ggcgcctgag ggcccgggat ggcgcgccat 12420 gcggccgcca tggtcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 12480 atctgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg 12540 cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatga gtattcaaca 12600 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 12660 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 12720 cgaactggat ctcaacagcg gtaagatcct tgagagttta cgccccgaag aacgttttcc 12780 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 12840 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 12900 agtcacagaa aagcatctca cggatggcat gacagtaaga gaattatgca gtgctgccat 12960 aaccatgagt gataacactg cggccaactt acttctggca acgatcggag gaccgaagga 13020 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 13080 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 13140 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 13200 aatagactgg atggaggcgg ataaagttgc aggatcactt ctgcgctcgg ccctcccggc 13260 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 13320 agcactgggg ccagatggta agccctcccg catcgtagtt atctacacga cggggagtca 13380 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 13440 ttggtaatga cagaagtcaa aagcctccgg tcggaggctt ttgactttct gctagatctg 13500 tttcaatgcg gtgaagggcc aggcagctgg ggattatgtc gagacccggc cagcatgttg 13560 gttttatcgc atattcagcg ttgtcgcgtt tacccaggta aaatggaagc agtgtatcgt 13620 ctgcgtgaat gtgcaaatca ggaacgtaac cgtggtacat agatgcagtc ccttgcgggt 13680 cgttcccttc aacgagtagg acgcggtgcc cttgcaaggc taaccattgc gcctggtgta 13740 ctgcagatga ggttttataa acccctccct tgtgtgacat aacggaaagt acaaccgggt 13800 ttttatcgtc aggtctttgg tttgggttac caaacacact ccgcatatgg ctaatttggt 13860 caattgtgta gccagcgcga cgttctactc ggcccctcat ctcaaaatca ggagccggta 13920 gacgaccagc tttttccgcg tctctgatag cctgcggtgt tacgccgatc aggtctgcaa 13980 cttctgttat accccagcgg cgagtaatac gacgcgcttc cgggctgtca tcgccgaact 14040 gtgcgatggc aatagcgcgc gtcatttcct gaccgcgatt gatacagtct ttcagcaaat 14100 taattaacga catcctgttt cctctcaaac atgcccttat ctttgtgttt ttcatcatac 14160 tttacgtttt taaagcaaag caacataaaa aaagcaaagt gacttagaaa acgcaaagtt 14220 aaggttcaaa tcaatttttt gatgcgctac agaagctatt tagcttcatc taagcgcaac 14280 ggtattactt acgttggtat atttaaaacc taacttaatg attttaaatg ataataaatc 14340 ataccaattg ctatcaaaag ttaagcgaac atgctgattt tcacgctgtt tatacacttt 14400 gaggcatctc tatctcttcc gtctctatat tgaaacacaa tcaaagaaca tcaatccatg 14460 tgacatcccc cactatctaa gaacaccata acagaacaca acataggaat gcaacattaa 14520 tgtatcaata attcggaaca tatgcactat atcatatctc aattacggaa catatcagca 14580 cacaattgcc cattatacgc 14600 <210> 4 <211> 14673 <212> DNA
<213> Artificial <220>
<223> NZASA
<400> 4 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 ggcgagaacg ggcagtggca gctgaagata gagacacctg caatcgtcct gcctgatgaa 1800 gaatccgttg agaccatcga cgaaccggat gatgagtccc aagacgacga gctggatgaa 1860 gatgaaattg agctcgacga gggtggcggc gatgaaccaa ccgaagagga agggccagaa 1920 gaacatcagc caactgctct aaaacccgtc ttcaagcctg caaaaaataa cggggacgga 1980 acgtacaaga tagagtttga atacgatgga aagcattatg cctggtccgg ccccgccgat 2040 agccctatgg ccgcaatgcg atccgcatgg gaaacgtact acagctaaaa gaaaagccac 2100 cggtgttaat cggtggcttt tttattgagg cctgtcccta cccatcccct gcaagggacg 2160 gaaggattag gcggaaactg cagctgcaac tacggacatc gccgtcccga ctgcagggac 2220 ttccccgcgt aaagcggggc ttaaattcgg gctggccaac cctatttttc tgcaatcgct 2280 ggcgatgtta gtttcgtgga tagcgtttcc agcttttcaa tggccagctc aaaatgtgct 2340 ggcagcacct tctccagttc cgtatcaata tcggtgatcg gcagctctcc acaagacata 2400 ctccggcgac cgccacgaac tacatcgcgc agcagctccc gttcgtagac acgcatgttg 2460 cccagagccg tttctgcagc cgttaatatc cggcgcagct cggcgatgat tgccgggaga 2520 tcatccacgg ttattgggtt cggtgatggg ttcctgcagg cgcggcggag agccatccag 2580 acgccgctaa cccatgcgtt acggtactga aaactttgtg ctatgtcgtt tatcaggccc 2640 cgaagttctt ctttctgccg ccagtccagt ggttcaccgg cgttcttagg ctcaggctcg 2700 acaaaagcat actcgccgtt tttccggata gctggcagaa cctcgttcgt cacccacttg 2760 cggaaccgcc aggctgtcgt cccctgtttc accgcgtcgc ggcagcggag gattatggtg 2820 tagagaccag attccgatac cacatttact tccctggcca tccgatcaag tttttgtgcc 2880 tcggttaaac cgagggtcaa tttttcatca tgatccagct tacgcaatgc atcagaaggg 2940 ttggctatat tcaatgcagc acagatatcc agcgccacaa accacgggtc accaccgaca 3000 agaaccaccc gtatagggtg gctttcctga aatgaaaaga cggagagagc cttcattgcg 3060 cctccccgga tttcagctgc tcagaaaggg acagggagca gccgcgagct tcctgcgtga 3120 gttcgcgcgc gacctgcaga agttccgcag cttcctgcaa atacagcgtg gcctcataac 3180 tggagatagt gcggtgagca gagcccacaa gcgcttcaac ctgcagcagg cgttcctcaa 3240 tcgtctccag caggccctgg gcgtttaact gaatctggtt catgcgatca cctcgctgac 3300 cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gagtcgacct gcaggcatgc aagcttcctg aatcgcccca tcatccagcc 10260 agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 10320 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 10380 tcaactcagc aaaagttcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat 10440 gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa 10500 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 10560 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 10620 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 10680 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 10740 cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 10800 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 10860 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 10920 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 10980 tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 11040 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 11100 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 11160 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 11220 atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg tttcccgttg 11280 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 11340 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct 11400 ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 11460 cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 11520 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggaagc 11580 ttgcatgcct gcaggtcgac tctagaggat ccccgagaac ccgataatcg ctaccagtga 11640 tgatggctgt tttgcggcgg cgtgagccat cggcaatttc gataatgcct gacgtccttc 11700 tggcgaacgc ggggttctgc tgtcctgaag tgaggaatga agggataagg tcggccagcg 11760 ctgattcgtt cagcaattcc tgatcacgtt cattaccgag ccaaaccatt gtggcctttt 11820 cgactttatc agcaggaatg gtttccagct taaaagtcac gttgcggcca tcaagcttga 11880 attcgtacgc agaaaggccc acccgaaggt gagccagtgt gattacattt gcggccgcat 11940 ttaaatgggc ccaatggccc gggaggccta cttaagtaag ccggcttagc tagcgggaca 12000 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc 12060 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga 12120 gcggacaaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctatttaggt 12180 gagactatag aatactcaag cttgcatgcg atacgtatcg ttaacgatgg atccgacgca 12240 cgtgcgaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt 12300 cgtgactggg aaaaccctgg cgtcacccaa cttaatcgcc ttgcagcaca tccccctttc 12360 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 12420 tgaatggcga atggcgcctg agctagcatt gccggcattc ttaagtaggc ctcccgggcc 12480 attggcgcgc catgcggccg ccatggtcaa ataaaacgaa aggctcagtc gaaagactgg 12540 gcctttcgtt ttaatctgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa 12600 gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa 12660 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 12720 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 12780 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt ttacgccccg 12840 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 12900 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 12960 ttgagtactc accagtcaca gaaaagcatc tcacggatgg catgacagta agagaattat 13020 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg gcaacgatcg 13080 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 13140 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 13200 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 13260 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggatca cttctgcgct 13320 cggccctccc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 13380 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgcatcgta gttatctaca 13440 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 13500 cactgattaa gcattggtaa tgacagaagt caaaagcctc cggtcggagg cttttgactt 13560 tctgctagat ctgtttcaat gcggtgaagg gccaggcagc tggggattat gtcgagaccc 13620 ggccagcatg ttggttttat cgcatattca gcgttgtcgc gtttacccag gtaaaatgga 13680 agcagtgtat cgtctgcgtg aatgtgcaaa tcaggaacgt aaccgtggta catagatgca 13740 , gtcccttgcg ggtcgttccc ttcaacgagt aggacgcggt gcccttgcaa ggctaaccat 13800 tgcgcctggt gtactgcaga tgaggtttta taaacccctc ccttgtgtga cataacggaa 13860 agtacaaccg ggtttttatc gtcaggtctt tggtttgggt taccaaacac actccgcata 13920 tggctaattt ggtcaattgt gtagccagcg cgacgttcta ctcggcccct catctcaaaa 13980 tcaggagccg gtagacgacc agctttttcc gcgtctctga tagcctgcgg tgttacgccg 14040 atcaggtctg caacttctgt tataccccag cggcgagtaa tacgacgcgc ttccgggctg 14100 tcatcgccga actgtgcgat ggcaatagcg cgcgtcattt cctgaccgcg attgatacag 14160 tctttcagca aattaattaa cgacatcctg tttcctctca aacatgccct tatctttgtg 14220 tttttcatca tactttacgt ttttaaagca aagcaacata aaaaaagcaa agtgacttag 14280 aaaacgcaaa gttaaggttc aaatcaattt tttgatgcgc tacagaagct atttagcttc 14340 atctaagcgc aacggtatta cttacgttgg tatatttaaa acctaactta atgattttaa 14400 atgataataa atcataccaa ttgctatcaa aagttaagcg aacatgctga ttttcacgct 14460 gtttatacac tttgaggcat ctctatctct tccgtctcta tattgaaaca caatcaaaga 14520 acatcaatcc atgtgacatc ccccactatc taagaacacc ataacagaac acaacatagg 14580 aatgcaacat taatgtatca ataattcgga acatatgcac tatatcatat ctcaattacg 14640 gaacatatca gcacacaatt gcccattata cgc 14673 <210> 5 <211> 14744 <212> DNA
<213> Artificial <220>
<223> NZAhd <400> 5 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 ggcgagaacg ggcagtggca gctgaagata gagacacctg caatcgtcct gcctgatgaa 1800 gaatccgttg agaccatcga cgaaccggat gatgagtccc aagacgacga gctggatgaa 1860 gatgaaattg agctcgacga gggtggcggc gatgaaccaa ccgaagagga agggccagaa 1920 gaacatcagc caactgctct aaaacccgtc ttcaagcctg caaaaaataa cggggacgga 1980 acgtacaaga tagagtttga atacgatgga aagcattatg cctggtccgg ccccgccgat 2040 agccctatgg ccgcaatgcg atccgcatgg gaaacgtact acagctaaaa gaaaagccac 2100 cggtgttaat cggtggcttt tttattgagg cctgtcccta cccatcccct gcaagggacg 2160 gaaggattag gcggaaactg cagctgcaac tacggacatc gccgtcccga ctgcagggac 2220 ttccccgcgt aaagcggggc ttaaattcgg gctggccaac cctatttttc tgcaatcgct 2280 ggcgatgtta gtttcgtgga tagcgtttcc agcttttcaa tggccagctc aaaatgtgct 2340 ggcagcacct tctccagttc cgtatcaata tcggtgatcg gcagctctcc acaagacata 2400 , ctccggcgac cgccacgaac tacatcgcgc agcagctccc gttcgtagac acgcatgttg 2460 cccagagccg tttctgcagc cgttaatatc cggcgcagct cggcgatgat tgccgggaga 2520 tcatccacgg ttattgggtt cggtgatggg ttcctgcagg cgcggcggag agccatccag 2580 acgccgctaa cccatgcgtt acggtactga aaactttgtg ctatgtcgtt tatcaggccc 2640 cgaagttctt ctttctgccg ccagtccagt ggttcaccgg cgttcttagg ctcaggctcg 2700 acaaaagcat actcgccgtt tttccggata gctggcagaa cctcgttcgt cacccacttg 2760 cggaaccgcc aggctgtcgt cccctgtttc accgcgtcgc ggcagcggag gattatggtg 2820 tagagaccag attccgatac cacatttact tccctggcca tccgatcaag tttttgtgcc 2880 tcggttaaac cgagggtcaa tttttcatca tgatccagct tacgcaatgc atcagaaggg 2940 ttggctatat tcaatgcagc acagatatcc agcgccacaa accacgggtc accaccgaca 3000 agaaccaccc gtatagggtg gctttcctga aatgaaaaga cggagagagc cttcattgcg 3060 cctccccgga tttcagctgc tcagaaaggg acagggagca gccgcgagct tcctgcgtga 3120 gttcgcgcgc gacctgcaga agttccgcag cttcctgcaa atacagcgtg gcctcataac 3180 tggagatagt gcggtgagca gagcccacaa gcgcttcaac ctgcagcagg cgttcctcaa 3240 tcgtctccag caggccctgg gcgtttaact gaatctggtt catgcgatca cctcgctgac 3300 cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 , tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gagtcgacct gcaggcatgc aagcttcctg aatcgcccca tcatccagcc 10260 agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 10320 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 10380 tcaactcagc aaaagttcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat 10440 gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa 10500 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 10560 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 10620 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 10680 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 10740 cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 10800 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 10860 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 10920 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 10980 tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 11040 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 11100 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 11160 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 11220 atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg tttcccgttg 11280 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 11340 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct 11400 ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 11460 cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 11520 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggaagc 11580 , ttgcatgcct gcaggtcgac tctagaggat ccccgagaac ccgataatcg ctaccagtga 11640 tgatggctgt tttgcggcgg cgtgagccat cggcaatttc gataatgcct gacgtccttc 11700 tggcgaacgc ggggttctgc tgtcctgaag tgaggaatga agggataagg tcggccagcg 11760 ctgattcgtt cagcaattcc tgatcacgtt cattaccgag ccaaaccatt gtggcctttt 11820 cgactttatc agcaggaatg gtttccagct taaaagtcac gttgcggcca tcaagcttga 11880 attcgtacgc agaaaggccc acccgaaggt gagccagtgt gattacattt gcggccagtc 11940 cagttacgct ggagtcacta gtgcggccgc gacaacttgt ctagggccca atggcccggg 12000 aggcctactt aagtaagccg gcttagctag cgggacaggt ttcccgactg gaaagcgggc 12060 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 12120 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gacaacaatt tcacacagga 12180 aacagctatg accatgatta cgccaagcta tttaggtgag actatagaat actcaagctt 12240 gcatgcgata cgtatcgtta acgatggatc cgacgcacgt gcgaattcgc cctatagtga 12300 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 12360 cacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 12420 ggcccgcacc gatcgccctt cccaacagtt gcgcagctga atggcgaatg gcgcctgagc 12480 tagcattgcc ggcattctta agtaggcctc ccgggccatt ggcgcgccat gacttgaagt 12540 cgcggccgca ctgaccattt aaatcatacc aacatggtca aataaaacga aaggctcagt 12600 cgaaagactg ggcctttcgt tttaatctga tcggcacgta agaggttcca actttcacca 12660 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa 12720 ggaagctaaa atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 12780 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 12840 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 12900 tttacgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 12960 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 13020 gaatgacttg gttgagtact caccagtcac agaaaagcat ctcacggatg gcatgacagt 13080 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 13140 ggcaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 13200 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 13260 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 13320 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggatc 13380 acttctgcgc tcggccctcc cggctggctg gtttattgct gataaatctg gagccggtga 13440 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgcatcgt 13500 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 13560 gataggtgcc tcactgatta agcattggta atgacagaag tcaaaagcct ccggtcggag 13620 gcttttgact ttctgctaga tctgtttcaa tgcggtgaag ggccaggcag ctggggatta 13680 tgtcgagacc cggccagcat gttggtttta tcgcatattc agcgttgtcg cgtttaccca 13740 ggtaaaatgg aagcagtgta tcgtctgcgt gaatgtgcaa atcaggaacg taaccgtggt 13800 acatagatgc agtcccttgc gggtcgttcc cttcaacgag taggacgcgg tgcccttgca 13860 aggctaacca ttgcgcctgg tgtactgcag atgaggtttt ataaacccct cccttgtgtg 13920 acataacgga aagtacaacc gggtttttat cgtcaggtct ttggtttggg ttaccaaaca 13980 cactccgcat atggctaatt tggtcaattg tgtagccagc gcgacgttct actcggcccc 14040 tcatctcaaa atcaggagcc ggtagacgac cagctttttc cgcgtctctg atagcctgcg 14100 gtgttacgcc gatcaggtct gcaacttctg ttatacccca gcggcgagta atacgacgcg 14160 cttccgggct gtcatcgccg aactgtgcga tggcaatagc gcgcgtcatt tcctgaccgc 14220 gattgataca gtctttcagc aaattaatta acgacatcct gtttcctctc aaacatgccc 14280 ttatctttgt gtttttcatc atactttacg tttttaaagc aaagcaacat aaaaaaagca 14340 aagtgactta gaaaacgcaa agttaaggtt caaatcaatt ttttgatgcg ctacagaagc 14400 tatttagctt catctaagcg caacggtatt acttacgttg gtatatttaa aacctaactt 14460 aatgatttta aatgataata aatcatacca attgctatca aaagttaagc gaacatgctg 14520 attttcacgc tgtttataca ctttgaggca tctctatctc ttccgtctct atattgaaac 14580 acaatcaaag aacatcaatc catgtgacat cccccactat ctaagaacac cataacagaa 14640 cacaacatag gaatgcaaca ttaatgtatc aataattcgg aacatatgca ctatatcata 14700 tctcaattac ggaacatatc agcacacaat tgcccattat acgc 14744 <210> 6 <211> 14549 <212> DNA
<213> Artificial <220>
<223> NZCK3 <400> 6 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 ggcgagaacg ggcagtggca gctgaagata gagacacctg caatcgtcct gcctgatgaa 1800 gaatccgttg agaccatcga cgaaccggat gatgagtccc aagacgacga gctggatgaa 1860 gatgaaattg agctcgacga gggtggcggc gatgaaccaa ccgaagagga agggccagaa 1920 gaacatcagc caactgctct aaaacccgtc ttcaagcctg caaaaaataa cggggacgga 1980 Ztard otgE Ec4-e54.4pqq ozepqE6opq 6p56.4q-eq46 poqeSqopep 6uoqp6qopE. qouqqqoaqq Teqqqqg 6q6Bpop361 6DP3q6P33e BgEoppppgq qq-equ.66465 pqlzeplppp ozLE 16oppEopu6 voElepopEo opqpqoppq6 66.6o6up6q3 Eqqqqabeo6 qp663oDEE.E.

6v6qq666.63 ;qpoqSqqpq 4DED6q6qq6 q8o6D66-qo6 OP3SUPP.2VP D'eOUDP.21q4 009E PBED606Teq 66Poqq.e.eqp opPoo6e6qo qP000DE614 EPP6DOPPOD 06600PPP6P
ovsE
PE.puoe.4663 opoPPogaT4 666eop6Poo o6pq66q566 6vqvq3q3r P6P5qq6v6u o8f7E povoopqpbq 655-eqvgbqg oTegooglup 61-el5Erqupe pEigggep6qg qpppoBlo&e.
oztE opEop66p6q Eopq61qqq.4 qp666qpqp6 6q.66p6.6-er 66666 Te6qp66pqo 09EE 6olDllo6p6 op6366qoE,e 6D883o66ov vppov66pEo ppEceopEqob .66ovqt,866o 00EE py6136pl33 yoqp6o61u3 qq661oTep6 gae-e13q6o5 66loop66Po 6epoloq6pq 017zE pvoq3plq63 Eibrobeobqo orpoqqD636 pp3epooBe6 Po6p6;66o6 g5t.qp5u55q ogTE
ove,1P3qoa6 .61.6o6vouTe vrobqooqqo Eceo63o3q6E. uSpo63o3v5 DEIDE.DEoqq5 ozTE
u.645361poq qoba6o6oDE va6p666-eae .666-2.esSpoq DE.qp5pDqq4 eBboopoqop 090E 636TIv3qqo 36v6e6p66o e6ee p6-4Dogq4D6 6T6E6E-Teq6 DOOPOOPPSU

pq665ospou uvoppobobe poquqpbrop 36u36qpp3q quqpqp6.6qq 0176z 6.66pp6eoTe D5q-ePo6oPq qp6PooqP6q solPoqq4.44 epD4.666-ebo 3eppqq.65o;
088z opEqBqqqqq beeoqeBooq soo66qoppq qopqqqpouo opqp6poqqe 6e6e6eq ozgz 6666 6E.6636vo66 o6oq6o5oop oqqqbqoppo q60.4.61o66p opEopp-ebBo 09[2 EclqOPOODPD q6o1163q33 Pp6eo65qp6 pq.26633q14 .116336313e 1P36PPPPOP

Elogobbeogo 66 J63 6.6pppo.;66 gEppoq6upo SpoSloqqqo qgo146u.e.BD
ot9z p3o65p3qpq ql6pq6qeqo 646-114pePp e6e66e qq6o6qppoo ppqa6opEop ogsz 6POOTe005P
6e5636E.D63 BE.Po5qopT4 5E6qp6q66p qq66EcTquqq 66epq ozsz P6p666336q iv6qp6o66o qo5e363563 oqugevqq6o obpo5qpqq; Boo6PEcepop ogtz 66e5e os6PqEoqq.6 opoloBvp&e, 0.6a6oTeopq aeR6OPOD60 DP5066DOqD

PqUOPEcePOP poqoqp6po6 5oge6.466D; vqPPoquq6o oqq6pooqoq qoopo6po66 0T7Ez qp5qEqp-epu oqobepoBBq ppo;;qqpbe opqqq6oBeg p554E.D434E, pqq6ge5o55 ogzz goboTepoBq pqqqqqpqop pypopE6gDE, 55pqq-ePpql 365663Bppy q6o6oppoql ozzz op666p36go pEopoqEopE, pqppuBEopq ov-235q36P3 6q3epp66o6 6pgge6EievE.
09-Ez 6op566-epa6 goopoqspoo vqopoqbqop 5.6p6qq.eggq qqqa6.6q66o queqq6455o ooTz opopEceuspE UPPPqD6PDP qouqEopuP6 56.4po600ge BoBTeroboo 664.eqopo6p otoz qu6006Doop 66pD4E6qop Bquqq-eobpp p6Erly6opqe pEqqqEreEceq p6evop46oy ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg cttaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacagatcg gttgcacggc 7860 tcagatgatt tctcgttaat ctggcgagcg acttccttca gccctctcag gctgtgcagg 7920 tcgttaaaat cgctgcattc cagctcaggg tcatcctcaa aagttgggta aacacattgg 7980 acgccggaaa acttctccat gatgtcgaat ccggtgcgga ggcctgtgtt gccttttcct 8040 tcagctgagg atttgcggtc gttatcgaga gcgcaagtga tttgcgcagc cgggtacatg 8100 ttcaccagct gctcgacaac gtgaatcatg ttgttagcgg aaaccgcaat gactaccgcg 8160 tcaaagcgtt ttttcgggtc gtttctggtc gccagccaga tggatgcccc ggtggcgaaa 8220 ccctctgcag tcgcaatttt ttgcgccccc tgcaggtcgc caataacaaa gcatgcaccg 8280 acgaaatcac cgttagtgat ggcgctggtc tggaacttgc caccattcag atcgatacgt 8340 tgccagccaa caatccgccc gtcttttctt ccgtccaggt gggacagagg tatcgccatg 8400 taagttgttg gtccacggct ccatttcgca ctgtcgtgac tggtcacgcg acgtatatca 8460 caagcgccaa atacgtcacg aattcccttt tttaccgcat aaggccagga gccatcttca 8520 gctggcgaat gttcccaggc gcgatggaaa gccaaccatc caagcaggcg ttcctgctcc 8580 atctgattgt tttttaaatc attaacgcgt tgttgttcag ctcggaggcg gcgtgcttca 8640 gcctggcgct ccatgcgtgc acgttcttct tccggctgag cgaccacggt cgcaccattc 8700 cgttgctgtt cacggcgata ctccgaaaac aggaatgaaa agccactcca ggagccagcg 8760 tcatgcgctt tttcaacgaa gttaacgaaa ggataactga tgccatcctt gctctgctca 8820 aggcgtgaat agatttccac acggccttta aggctcttct gcagagcttc cggggaggaa 8880 ttattgtagg tggtatagcg ctctacacca ccgcgcggat tgagctgaat cttatcagca 8940 cacgcaggcc agttgatacc ggccatcttc gccagctcag tcagctcatc acgtgccgcg 9000 tcaagcagtg aaaacggatc gctgccaaag cgctccgcgt agaattcttg taaggtcatt 9060 ttttagcctt tccatgcgaa ttagcatttt ttcgggttga aaaaatccgc aggagcagcc 9120 acaataaacg cactatcttt ctgaaggacg tatctgcgtt atcgtggcta cttcctgaaa 9180 aaggcccgag tttgccgact cgggtttttt ttcgtctttt ttcggctgct acggtctggt 9240 tcaaccccga caaagtatag atcggattaa accagaatta tagtcagcaa taaaccctgt 9300 tattgtatca tctaccctca accatgaacg atttgatcgt accgactact tggtgcacaa 9360 attgaagatc acttttatca tggataaccc gttgagagtt agcactatca aggtagtaat 9420 gctgctcgtc ataacgggct aatcgttgaa ttgtgatctc gccgttatta tcacaaacca 9480 gtacatcctc acccggtaca agcgtaagtg aagaatcgac caggataacg tctcccggct 9540 ggtagtttcg ctgaatctgg ttcccgaccg tcagtgcgta aacggtgttc cgttgactca 9600 cgaacggcag gaatcgctct gtgttggcag gttctccagg ctgccagtct ctatccggtc 9660 cggtctctgt cgtaccaata acaggaacgc ggtctggatc agattcagtg ccatacagta 9720 tccattgcac gggcttacgc aggcattttg ccagcgatag cccgatctcc agcgacggca 9780 tcacgtcgcc acgttctaag ttttggacgc ccggaagaga gattcctaca gcttctgcca 9840 cttgcttcag cgtcagtttc agctctaaac ggcgtgcttt cagtcgttcg cctcgtgttt 9900 tcataccctt aatcataaat gatctcttta tagctggcta taatttttat aaattatacc 9960 tagctttaat tttcacttat tgattataat aatccccatg aaacccgaag aacttgtgcg 10020 ccatttcggc gatgtggaaa aagcagcggt tggcgtgggc gtgacacccg gcgcagtcta 10080 tcaatggctg caagctgggg agattccacc tctacgacaa agcgatatag aggtccgtac 10140 cgcgtacaaa ttaaagagtg atttcacctc tcagcgcatg ggtaaggaag ggcataacag 10200 gggatcctct agagtcgacc tgcaggcatg caagcttcct gaatcgcccc atcatccagc 10260 cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca gttggtgatt 10320 ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc 10380 ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa 10440 tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca 10500 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt 10560 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc 10620 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa 10680 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa 10740 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat 10800 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc 10860 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg 10920 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg 10980 ttttccctgg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct 11040 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa 11100 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc 11160 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc 11220 catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt 11280 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc 11340 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc 11400 tttgttgaat aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca 11460 acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca 11520 aagctctcat caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggaag 11580 cttgcatgcc tgcaggtcga ctctagagga tccccgagaa cccgataatc gctaccagtg 11640 atgatggctg ttttgcggcg gcgtgagcca tcggcaattt cgataatgcc tgacgtcctt 11700 ctggcgaacg cggggttctg ctgtcctgaa gtgaggaatg aagggataag gtcggccagc 11760 gctgattcgt tcagcaattc ctgatcacgt tcattaccga gccaaaccat tgtggccttt 11820 tcgactttat cagcaggaat ggtttccagc ttaaaagtca cgttgcggcc atcaagcttg 11880 aattcgtacg cagaaaggcc cacccgaagg tgagccagtg tgattacatt tgcggccagt 11940 ccagttacgc tggagtcact agtgcggccg cgacaacttg tctagggccc aatggcccgg 12000 gaggcctact taagtaagcc ggcttagcta gcgggacagg tttcccgact ggaaagcggg 12060 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 12120 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggacaacaat ttcacacagg 12180 aaacagctat gaccatgatt acgccaagct atttaggtga gactatagaa tactcaagct 12240 tgcatgcgat acgtatcgtt aacgatggat ccgacgcacg tgcgaattcg ccctatagtg 12300 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 12360 tcacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 12420 aggcccgcac cgatcgccct tcccaacagt tgcgcagctg aatggcgaat ggcgcctgag 12480 ctagcattgc cggcattctt aagtaggcct cccgggccat tggcgcgcca gacttgaagt 12540 cgcggccgca ctgaccattt aaatcatacc aacatggtca aataaaacga aaggctcagt 12600 cgaaagactg ggcctttcgt tttaatctga tcggcacgta agaggttcca actttcacca 12660 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa 12720 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca 12780 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt 12840 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc 12900 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc gtatggcaat 12960 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga 13020 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct 13080 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg 13140 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga 13200 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta 13260 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 13320 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg 13380 cggggcgtaa cctaggtgac agaagtcaaa agcctccggt cggaggcttt tgactttctg 13440 ctagatctgt ttcaatgcgg tgaagggcca ggcagctggg gattatgtcg agacccggcc 13500 agcatgttgg ttttatcgca tattcagcgt tgtcgcgttt acccaggtaa aatggaagca 13560 gtgtatcgtc tgcgtgaatg tgcaaatcag gaacgtaacc gtggtacata gatgcagtcc 13620 cttgcgggtc gttcccttca acgagtagga cgcggtgccc ttgcaaggct aaccattgcg 13680 cctggtgtac tgcagatgag gttttataaa cccctccctt gtgtgacata acggaaagta 13740 caaccgggtt tttatcgtca ggtctttggt ttgggttacc aaacacactc cgcatatggc 13800 taatttggtc aattgtgtag ccagcgcgac gttctactcg gcccctcatc tcaaaatcag 13860 gagccggtag acgaccagct ttttccgcgt ctctgatagc ctgcggtgtt acgccgatca 13920 ggtctgcaac ttctgttata ccccagcggc gagtaatacg acgcgcttcc gggctgtcat 13980 cgccgaactg tgcgatggca atagcgcgcg tcatttcctg accgcgattg atacagtctt 14040 tcagcaaatt aattaacgac atcctgtttc ctctcaaaca tgcccttatc tttgtgtttt 14100 tcatcatact ttacgttttt aaagcaaagc aacataaaaa aagcaaagtg acttagaaaa 14160 cgcaaagtta aggttcaaat caattttttg atgcgctaca gaagctattt agcttcatct 14220 aagcgcaacg gtattactta cgttggtata tttaaaacct aacttaatga ttttaaatga 14280 taataaatca taccaattgc tatcaaaagt taagcgaaca tgctgatttt cacgctgttt 14340 atacactttg aggcatctct atctcttccg tctctatatt gaaacacaat caaagaacat 14400 caatccatgt gacatccccc actatctaag aacaccataa cagaacacaa cataggaatg 14460 caacattaat gtatcaataa ttcggaacat atgcactata tcatatctca attacggaac 14520 atatcagcac acaattgccc attatacgc 14549 <210> 7 <211> 12873 <212> DNA
<213> Artificial <220>

<223> NZTC2 <400> 7 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 0c3kri 009E pEpo6o6.1.2.4 56E 3ppopEu6qo qpooap55T3 .6-eu5pouppo 066DDPPPEP
otsE u6ppop;66o oeoppoqDqq 688pop6poo DEpq.65.4666 Suqpqoqoqp P6p6T3SPEce oeT7E opPoDploBq .666uTeq6qq oqPqopqq-ep 6.4pq6EcTeop pEcTlqp.26.4.4 qp-epo&IDEP
oztE Dp8oPEED6q E.Doq6.4.4qqg qp666.4-eqp6 6.466DE6-epp 6Ec4E6qp-eD5 qp6qp6.6pqo 09EE ED.431138p6 Dp6o66qopp 6o66qo6EDP PppopEE.P.63 pp6Pou6qp6 55666D
00EE op6q36Doo po.quEcEqpo .6613-1-ep6 1Dup;qq6o6 66-133366e3 6poogpl6o'l 017zE ppoqpagEo 5Eceo6ppEc;o ouPollo638 puppoopEce6 ep6e.6q66o6 16p4PEE.664 08TE opuqpoqopb 615a6pouqp ppoSqoplqo 6po6poqq5p p6po6qoppE, o6D6pEoqq5 oz-E p61.63.6q33l .1a6p8o6DD6 pa6p666po-e 666e5ceol D6.1D6po p6600Do-loo oe0E Ea6.44-epqo 36P6a6P660 P6UPPPEc3PP ubqopqq4D5 63666-equq6 DOOPDOPPBP
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0861 p6or66.66o PPTePPPPU3 6q006PPOql 0q6DOOPPPP qpqa6qaePo DEPOTeOPPE
0z6.1 pa6upp666E, .266p6uu6op PPOOPPETe6 D65366'4666 p6orbow6p 666 0981 up.6.4P66;o6 P6DP6OPEEP DOD16P5TE6 qu6600Pp6o P6DT2DOPEP 666 0081 up8T25-4336 woq6oqppo 5goopop6p6 pqp6pp6qp6 po65q6po65 66666 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga -7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctkaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gacgcagaaa ggcccacccg aaggtgagcc agtgtgatta catttgcggc 10260 cagtccagtt acgctggagt cactagtgcg gccgcgacaa cttgtctagg gcccaatggc 10320 ccgggaggcc tacttaagta agccggctta gctagcggga caggtttccc gactggaaag 10380 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 10440 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggacaa caatttcaca 10500 caggaaacag ctatgaccat gattacgcca agctatttag gtgagactat agaatactca 10560 agcttgcatg cgatacgtat cgttaacgat ggatccgacg cacgtgcgaa ttcgccctat 10620 agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 10680 ggcgtcaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc 10740 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gctgaatggc gaatggcgcc 10800 tgagctagca ttgccggcat tcttaagtag gcctcccggg ccattggcgc gccagacttg 10860 aagtcgcggc cgcactgacc atttaaatca taccaacatg gtcaaataaa acgaaaggct 10920 , cagtcgaaag actgggcctt tcgttttaat ctgatcggca cgtaagaggt tccaactttc 10980 accataatga aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag 11040 ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat 11100 ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga 11160 ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt 11220 atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg 11280 caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc 11340 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt 11400 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta 11460 aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt 11520 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat 11580 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt 11640 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc 11700 agggcggggc gtaacctagg tgacagaagt caaaagcctc cggtcggagg cttttgactt 11760 tctgctagat ctgtttcaat gcggtgaagg gccaggcagc tggggattat gtcgagaccc 11820 ggccagcatg ttggttttat cgcatattca gcgttgtcgc gtttacccag gtaaaatgga 11880 agcagtgtat cgtctgcgtg aatgtgcaaa tcaggaacgt aaccgtggta catagatgca 11940 gtcccttgcg ggtcgttccc ttcaacgagt aggacgcggt gcccttgcaa ggctaaccat 12000 tgcgcctggt gtactgcaga tgaggtttta taaacccctc ccttgtgtga cataacggaa 12060 agtacaaccg ggtttttatc gtcaggtctt tggtttgggt taccaaacac actccgcata 12120 tggctaattt ggtcaattgt gtagccagcg cgacgttcta ctcggcccct catctcaaaa 12180 tcaggagccg gtagacgacc agctttttcc gcgtctctga tagcctgcgg tgttacgccg 12240 atcaggtctg caacttctgt tataccccag cggcgagtaa tacgacgcgc ttccgggctg 12300 tcatcgccga actgtgcgat ggcaatagcg cgcgtcattt cctgaccgcg attgatacag 12360 tctttcagca aattaattaa cgacatcctg tttcctctca aacatgccct tatctttgtg 12420 tttttcatca tactttacgt ttttaaagca aagcaacata aaaaaagcaa agtgacttag 12480 aaaacgcaaa gttaaggttc aaatcaattt tttgatgcgc tacagaagct atttagcttc 12540 atctaagcgc aacggtatta cttacgttgg tatatttaaa acctaactta atgattttaa 12600 atgataataa atcataccaa ttgctatcaa aagttaagcg aacatgctga ttttcacgct 12660 gtttatacac tttgaggcat ctctatctct tccgtctcta tattgaaaca caatcaaaga 12720 acatcaatcc atgtgacatc ccccactatc taagaacacc ataacagaac acaacatagg 12780 aatgcaacat taatgtatca ataattcgga acatatgcac tatatcatat ctcaattacg 12840 gaacatatca gcacacaatt gcccattata cgc 12873 <210> 8 <211> 22 <212> DNA
<213> Artificial <220>
<223> T7-RC-Del <400> 8 acgcagaaag gcccacccga ag 22 <210> 9 <211> 18 <212> DNA
<213> Artificial <220>
<223> pCmOR
<400> 9 tttagcttcc ttagctcc 18 <210> 10 <211> 23 <212> DNA
<213> Artificial <220>
<223> NZg7847a-F2 <400> 10 agatcggttg cacggctcag atg 23 <210> 11 <211> 49 <212> DNA
<213> Artificial <220>
<223> NZg7847a-F3 <400> 11 agatcggttg cacggctcag atgatttctc gttaactggc gagcgactt 49 <210> 12 <211> 74 <212> DNA
<213> Artificial <220>

<223> NZ-RevB
<400> 12 gccgcttgac ttcaagtcta atggcccggg aggcctactt aagattcgcc attcagctgc 60 gcaactgttg ggaa 74 <210> 13 <211> 74 <212> DNA
<213> Artificial <220>
<223> NZ-RevA
<400> 13 aaatggtcag ttaatcagtt ctatgtacca gcaaggtcca gttgtaagcg gccgcttgac 60 ttcaagtcta atgg 74 <210> 14 <211> 22 <212> DNA
<213> Artificial <220>
<223> NZ-RevC
<400> 14 aaatggtcag ttaatcagtt ct 22 <210> 15 <211> 7260 <212> DNA
<213> Artificial <220>
<223> pEZ BAC
<400> 15 aaggaatatt cagcaatttg cccgtgccga agaaaggccc acccgtgaag gtgagccagt 60 gagttgattg ctacgtaaat aacttcgtat agcatacatt atacgaagtt atggactagg 120 cgcgccagaa gagagaaaga aggaaagcgg ccgcacaggt ttcccgactg gaaagcgggc 180 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 240 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 300 aacagctatg accatgatta cgccaagcta tttaggtgag actatagaat actcaagctt 360 gcatgcgata cgtatcgtta acgatggatc cgacgcacgt gcgaattcgc cctatagtga 420 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 480 cacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 540 ggcccgcacc gatcgccctt cccaacagtt gcgcagctga atggcgaatg gcgcctgatg 600 cggtattttc tccttacggc ggccgcttga cataacttcg tatagcatac attatacgaa 660 gttatgttta aacattagca gaaagtcaaa ggcctccggt cggaggcttt tgactaaaac 720 ttcccttggg gttatcattg ggccgagacc gcctgaagag gacttccatt gttcattcca 780 cggacaaaaa cagagaaagg aaacgacaga ggccaaaaag ctcgctttca gcacctgtcg 840 tttcctttct tttcagaggg tattttaaat aaaaacatta agttatgacg aagaagaacg 900 gaaacgcctt aaaccggaaa attttcataa atagcgaaaa cccgcgaggt cgccgccccg 960 taacctgtcg gatcaccgga aaggacccgt aaagtgataa tgattatcat ctacatatca 1020 caacgtgcgt ggaggccatc aaaccacgtc aaataatcaa ttatgacgca ggtatcgtat 1080 taattgatct gcatcaactt aacgtaaaag caacttcaga caatacaaat cagcgacact 1140 gaatacgggg caacctcatg tcgcctgaag agtgagaccg gccctgatcg gcacgtaaga 1200 ggctccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg 1260 agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt 1320 gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt 1380 acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat 1440 aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg 1500 gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt 1560 tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac 1620 gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg 1680 gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg 1740 agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc 1800 accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt 1860 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac 1920 tgcgatgagt ggcagggcgg ggcgtaaaaa tgtaatcacc tggctcacct tcgggtgggc 1980 ctttcacact tgcatcggat gcagcccggt gaacgtgccg gcacggcctg ggtaaccagg 2040 tattttgtcc acataaccgt gcgcaaaatg ttgtggataa gcaggacaca gcagcaatcc 2100 acagcaggca tacaaccgca caccgaggtt actccgttct acaggttacg acgacatgtc 2160 aatacttgcc cttgacaggc attgatggaa tcgtagtctc acgctgatag tctgatcgac 2220 aatacaagtg ggaccgtggt cccagaccga taatcagacc gacaacacga gtgggatcgt 2280 ggtcccagac taataatcag accgacgata cgagtgggac cgtggtccca gactaataat 2340 cagaccgacg atacgagtgg gaccgtggtt ccagactaat aatcagaccg acgatacgag 2400 , tgggaccgtg gtcccagact aataatcaga ccgacgatac gagtgggacc atggtcccag 2460 actaataatc agaccgacga tacgagtggg accgtggtcc cagtctgatt atcagaccga 2520 cgatacgagt gggaccgtgg tcccagacta ataatcagac cgacgatacg agtgggaccg 2580 tggtcccaga ctaataatca gaccgacgat acgagtggga ccgtggtccc agtctgatta 2640 tcagaccgac gatacaagtg gaacagtggg cccagagaga atattcaggc cagttatgct 2700 ttctggcctg taacaaagga cattaagtaa agacagataa acgtagacta aaacgtggtc 2760 gcatcagggt gctggctttt caagttcctt aagaatggcc tcaattttct ctatacactc 2820 agttggaaca cgagacctgt ccaggttaag caccatttta tcgcccttat acaatactgt 2880 cgctccagga gcaaactgat gtcgtgagct taaactagtt cttgatgcag atgacgtttt 2940 aagcacagaa gttaaaagag tgataacttc ttcagcttca aatatcaccc cagctttttt 3000 ctgctcatga aggttagatg cctgctgctt aagtaattcc tctttatctg taaaggcttt 3060 ttgaagtgca tcacctgacc gggcagatag ttcaccgggg tgagaaaaaa gagcaacaac 3120 tgatttaggc aatttggcgg tgttgataca gcgggtaata atcttacgtg aaatattttc 3180 cgcatcagcc agcgcagaaa tatttccagc aaattcattc tgcaatcggc ttgcataacg 3240 ctgaccacgt tcataagcac ttgttgggcg ataatcgtta cccaatctgg ataatgcagc 3300 catctgctca tcatccagct cgccaaccag aacacgataa tcactttcgg taagtgcagc 3360 agctttacga cggcgactcc catcggcaat ttctatgaca ccagatactc ttcgaccgaa 3420 cgccggtgtc tgttgaccag tcagtagaaa agaagggatg agatcatcca gtgcgtcctc 3480 agtaagcagc tcctggtcac gttcattacc tgaccatacc cgagaggtct tctcaacact 3540 atcaccccgg agcacttcaa gagtaaactt cacatcccga ccacatacag gcaaagtaat 3600 ggcattaccg cgagccatta ctcctacgcg cgcaattaac gaatccacca tcggggcagc 3660 tggtgtcgat aacgaagtat cttcaaccgg ttgagtattg agcgtatgtt ttggaataac 3720 aggcgcacgc ttcattatct aatctcccag cgtggtttaa tcagacgatc gaaaatttca 3780 ttgcagacag gttcccaaat agaaagagca tttctccagg caccagttga agagcgttga 3840 tcaatggcct gttcaaaaac agttctcatc cggatctgac ctttaccaac ttcatccgtt 3900 tcacgtacaa cattttttag aaccatgctt ccccaggcat cccgaatttg ctcctccatc 3960 cacggggact gagagccatt actattgctg tatttggtaa gcaaaatacg tacatcaggc 4020 tcgaaccctt taagatcaac gttcttgagc agatcacgaa gcatatcgaa aaactgcagt 4080 gcggaggtgt agtcaaacaa ctcagcaggc gtgggaacaa tcagcacatc agcagcacat 4140 acgacattaa tcgtgccgat acccaggtta ggcgcgctgt caataactat gacatcatag 4200 , tcatgagcaa cagtttcaat ggccagtcgg agcatcaggt gtggatcggt gggcagttta 4260 ccttcatcaa atttgcccat taactcagtt tcaatacggt gcagagccag acaggaagga 4320 ataatgtcaa gccccggcca gcaagtgggc tttattgcat aagtgacatc gtccttttcc 4380 ccaagataga aaggcaggag agtgtcttct gcatgaatat gaagatctgg tacccatccg 4440 tgatacattg aggctgttcc ctgggggtcg ttaccttcca cgagcaaaac acgtagcccc 4500 ttcagagcca gatcctgagc aagatgaaca gaaactgagg ttttgtaaac gccaccttta 4560 tgggcagcaa ccccgatcac cggtggaaat acgtcttcag cacgtcgcaa tcgcgtacca 4620 aacacatcac gcatatgatt aatttgttca attgtataac caacacgttg ctcaacccgt 4680 cctcgaattt ccatatccgg gtgcggtagt cgccctgctt tctcggcatc tctgatagcc 4740 tgagaagaaa ccccaactaa atccgctgct tcacctattc tccagcgccg ggttattttc 4800 ctcgcttccg ggctgtcatc attaaactgt gcaatggcga tagccttcgt catttcatga 4860 ccagcgttta tgcactggtt aagtgtttcc atgagtttca ttctgaacat cctttaatca 4920 ttgctttgcg tttttttatt aaatcttgca atttactgca aagcaacaac aaaatcgcaa 4980 agtcatcaaa aaaccgcaaa gttgtttaaa ataagagcaa cactacaaaa ggagataaga 5040 agagcacata cctcagtcac ttattatcac tagcgctcgc cgcagccgtg taaccgagca 5100 tagcgagcga actggcgagg aagcaaagaa gaactgttct gtcagatagc tcttacgctc 5160 agcgcaagaa gaaatatcca ccgtgggaaa aactccaggt agaggtacac acgcggatag 5220 ccaattcaga gtaataaact gtgataatca accctcatca atgatgacga actaaccccc 5280 gatatcaagt cacatgacga agggaaagag aaggaaatca actgtgacaa actgccctca 5340 aatttggctt ccttaaaaat tacagttcaa aaagtatgag aaaatccatg caggctgaag 5400 gaaacagcaa aactgtgaca aattaccctc agtaggtcag aacaaatgtg acgaaccacc 5460 ctcaaatctg tgacagataa ccctcagact atcctgtcgt catggaagtg atatcgcgga 5520 aggaaaatac gatatgagtc gtctggcggc ctttcttttt ctcaatgtat gagaggcgca 5580 ttggagttct gctgttgatc tcattaacac agacctgcag gaagcggcgg cggaagtcag 5640 gcatacgctg gtaactttga ggcagctggt aacgctctat gatccagtcg attttcagag 5700 agacgatgcc tgagccatcc ggcttacgat actgacacag ggattcgtat aaacgcatgg 5760 catacggatt ggtgatttct tttgtttcac taagccgaaa ctgcgtaaac cggttctgta 5820 acccgataaa gaagggaatg agatatgggt tgatatgtac actgtaaagc cctctggatg 5880 gactgtgcgc acgtttgata aaccaaggaa aagattcata gcctttttca tcgccggcat 5940 cctcttcagg gcgataaaaa accacttcct tccccgcgaa actcttcaat gcctgccgta 6000 tatccttact ggcttccgca gaggtcaatc cgaatatttc agcatattta gcaacatgga 6060 tctcgcagat accgtcatgt tcctgtaggg tgccatcaga ttttctgatc tggtcaacga 6120 acagatacag catacgtttt tgatcccggg agagactata tgccgcctca gtgaggtcgt 6180 ttgactggac gattcgcggg ctatttttac gtttcttgtg attgataacc gctgtttccg 6240 ccatgacaga tccatgtgaa gtgtgacaag tttttagatt gtcacactaa ataaaaaaga 6300 gtcaataagc agggataact ttgtgaaaaa acagcttctt ctgagggcaa tttgtcacag 6360 ggttaagggc aatttgtcac agacaggact gtcatttgag ggtgatttgt cacactgaaa 6420 gggcaatttg tcacaacacc ttctctagaa ccagcatgga taaaggccta caaggcgctc 6480 taaaaaagaa gatctaaaaa ctataaaaaa aataattata aaaatatccc cgtggataag 6540 tggataaccc caagggaagt tttttcaggc atcgtgtgta agcagaatat ataagtgctg 6600 ttccctggtg cttcctcgct cactcgaccg ggagggttcg agaagggggg gcacccccct 6660 tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg tttataaata 6720 ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg cggaaaccct 6780 tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg tgcgcccctc 6840 atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg tcagtagtcg 6900 cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac atcatctgtg 6960 ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag ctccacgtcg 7020 ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag tcggcccctc 7080 aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg aggtatccac 7140 aacgccggcg gccggccgcg gtgtctcgca cacggcttcg acggcgtttc tggcgcgttt 7200 gcagggccat agacggccgc cagcccagcg gcgagggcaa ccagcccggt gagcgtcgga 7260 <210> 16 <211> 55 <212> DNA
<213> Artificial <220>
<223> T7RC NotF
<400> 16 acgcagaaag gcccacccga aggtgagcca gtgtgattac atttgcggcc gcatt 55 <210> 17 <211> 69 <212> DNA
<213> Artificial <220>
<223> NSAS-LacZ-F

<400> 17 gtgtgattac atttgcggcc gcatttaaat gggcccggga caggtttccc gactggaaag 60 cgggcagtg 69 <210> 18 <211> 70 <212> DNA
<213> Artificial <220>
<223> NNASA-LacZ-R
<400> 18 cttgttttat ttgaccatgg cggccgcatg gcgcgccatc ccgggccctc aggcgccatt 60 cgccattcag 70 <210> 19 <211> 21 <212> DNA
<213> Artificial <220>
<223> rrn-Fd <400> 19 tgcggccgcc atggtcaaat a 21 <210> 20 <211> 93 <212> DNA
<213> Artificial <220>
<223> rrn-pCmF2 <400> 20 tgcggccgcc atggtcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 60 aatctgatcg gcacgtaaga ggttccaact ttc 93 <210> 21 <211> 55 <212> DNA
<213> Artificial <220>
<223> TonAmpR
<400> 21 tccgaccgga ggcttttgac ttctgtcatt accaatgctt aatcagtgag gcacc 55 <210> 22 <211> 58 <212> DNA
<213> Artificial <220>
<223> TonB-R
<400> 22 ttgaaacaga tctagcagaa agtcaaaagc ctccgaccgg aggcttttga cttctgtc 58 <210> 23 <211> 22 <212> DNA
<213> Artificial <220>
<223> T7del <400> 23 acgcagaaag gcccacccga ag 22 <210> 24 <211> 87 <212> DNA
<213> Artificial <220>
<223> TonBR2 <400> 24 attgaaacag atctagcaga aagtcaaaag cctccgaccg gaggcttttg acttctgtca 60 ttaccaatgc ttaatcagtg aggcacc 87 <210> 25 <211> 48 <212> DNA
<213> Artificial <220>
<223> LacANN-For <400> 25 tacttaagta agccggctta gctagcggga caggtttccc gactggaa 48 <210> 26 <211> 49 <212> DNA
<213> Artificial <220>
<223> LacANN-Rev <400> 26 acttaagaat gccggcaatg ctagctcagg cgccattcgc cattcagct 49 <210> 27 <211> 47 <212> DNA
<213> Artificial <220>
<223> LacApSA-For <400> 27 ttatatgggc ccaatggccc gggaggccta cttaagtaag ccggctt 47 <210> 28 <211> 50 <212> DNA
<213> Artificial <220>
<223> LacAsSA-Rev <400> 28 aatagttggc gcgccaatgg cccgggaggc ctacttaaga atgccggcaa 50 <210> 29 <211> 78 <212> DNA
<213> Artificial <220>
<223> LacE-SL1-F
<400> 29 taactgtggc cagtccagtt acgctggagt cactagtgcg gccgcgacaa cttgtctagg 60 gcccaatggc ccgggagg 78 <210> 30 <211> 70 <212> DNA
<213> Artificial <220>
<223> LacA SR2-Rev <400> 30 ctaggaacat gttggtatga tttaaatggt cagtgcggcc gcgacttcaa gtctggcgcg 60 ccaatggccc 70 <210> 31 <211> 39 <212> DNA
<213> Artificial <220>
<223> 7847-F2 <400> 31 agatcggttg cacggctcag atgatttctc gttaactgg 39 <210> 32 <211> 81 <212> DNA
<213> Artificial <220>
<223> CamTonB-Rev <400> 32 gaaacagatc tgatctagca gaaagtcaaa agcctccgac cggaggcttt tgacttctgt 60 cacctaggtt acgccccgcc c 81 <210> 33 <211> 41 <212> DNA
<213> Artificial <220>
<223> Primer 1 <400> 33 ttcttatggc cagggaggcc gctctgggta taagcgtaag g 41 <210> 34 <211> 41 <212> DNA
<213> Artificial <220>
<223> Primer 2 <400> 34 aactagtggc cagggaggcc atcagccagg cgacgaatca g 41 <210> 35 <211> 41 <212> DNA
<213> Artificial <220>
<223> Primer 3 <400> 35 ggacttgggc cacccaggcc ttgtaaatgc agtatggatt g 41 <210> 36 <211> 42 <212> DNA
<213> Artificial <220>
<223> Primer 4 <400> 36 atcctagggc cacccaggcc agatattgga gagttgcacc ag 42 <210> 37 <211> 14737 <212> DNA
<213> Artificial <220>
<223> pNZ-Sfi <400> 37 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 L90fud ocIEE
Dp6o,o6Dqoo uoqp6oEcTeo qq66qoTep5 qoppqqq6DE ESqopo56p3 E63.4 otzE
ppo3oqq63 66P36uDE.qo OPPDT4D5O6 PPOPDDD5P5 PD6p6.466D5 48Pqu6u66.4 081E op-equoqp36 546DEcepuTe ppo8qopqqo BpoBooqq5e. p6eo6qoppE obo6p6oqq6 ozTE
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ogsz BPDOTeDDEP 5pEE.066363 66-ep5qopqg 666quE46ED qq6E.6.44-eqq. HopopTepq ozsz P6p666o36.4 qp6T26366o qD6p36p66D pqPquuqq6D DEppEc4Dqqq 6006p6poop 09T7E Eqq.64-e3Eae opEcegEoq46 popqp6p3Bp p6o6Dqpopq DPP6OPOD.60 Da6066D0q0 00f7Z
PTeOPEPPOU DD4DqD6po6 6oqp6gE6pq pTepoquqbp oqq6uppqpq goopp5p365 0T7Ez qp6.46qpppp oqp6poo66.4 ppoqqqqp6p Doqqq6o6pq P66q6pqqqE, uqq6qu6366 08zz q36o.T2po62 oqqqqqpqpo opppoS6qoE, 66oqq-eppqq DE6663Supp T6DB000pqq ozzz opEEEpo6q3 p600pq6Do6 oquDp66opq opppbqpEcep 6qoppp6606 6-eq;p66uu6 091z 6op666-epo6 qoppoqppoo pqoppqagoo 66p6qqpq11 qqq.DEBT6ED Tee-4-46-466o opiz opopEpppub UPPPqD6PDP qopq6oppe6 5EquD5DoTe 6o6Tepp5oo 656p of7oz TeSopEcoop 66opq66qop 8qpqqppEcep p66quEopqp e6qqq6pEpq p6PPOPqE1OP

p6op6.66.6o PPTPPPPPPD Eq.DOSPPOqq. Dq6DOOPPPP qoqp6qoppo obeoTeopp6 0z61 pp6poo6E6p PEEP6EU5OD PUDDPPEcTeE, 366368q666 uEcuEtjqoBu 6qqrupEc4p6 pu6TE,66.436 PBOPSOPEPP opoq6pETE6 Te6600ppSo P5OTeDDP6P 66DaTea6 0081 u-e6e6qoo6 ;ooq5o4PPo EqDopop6p6 P-Te6P-e6;p6 pD66;EPD6E EoPPEce6D66 otLi TlEolqEceo6 666;TeDETe 6w6o064.4.4 6eE6qoppq6 Eco5uqqa6q PEop63336u 0891 ;-1qppp.T1-43 DE,E,Epopqp PD6POPPOOP P1PPUPPO5P oqp000pbae DEce66.466qo EppEcepqm63 oppp6q-epoq oq6opq6o66 qa6op6T6Ece 5p 5r,5 EPODETErea6 0981 Te6op66qop uubppEcloqo 55q55q368E, DOPOPPPP8q p6666qq6pp 6.4pop5o55q , cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagccctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg ctgaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacaggtcg gttgcacggc 7860 tcagatgatt tctcgttaac tggcgagcga cttccttcag ccctctcagg ctgtgcaggt 7920 cgttaaaatc gctgcattcc agctcagggt catcctcaaa agttgggtaa acacatttga 7980 cgccggaaaa cttctccatg atgtcgaatc cggtgcggag gcctgtgttg ccttttcctt 8040 cagctgagga tttgcggtcg ttatcgagag cgcaagtgat ttgcgcagcc gggtacatgt 8100 tcaccagctg ctcgacaacg tgaatcatgt tgttagcgga aaccgcaatg actaccgcgt 8160 caaagcgttt tttcgggtcg tttctggtcg ccagccagat ggatgccccg gtggcgaaac 8220 cctctgcagt cgcaattttt tgcgccccct gcaggtcgcc aataacaaag catgcaccga 8280 cgaaatcacc gttagtgatg gcgctggtct ggaacttgcc accattcaga tcgatacgtt 8340 gccagccaac aatccgcccg tcttttcttc cgtccaggtg ggacagaggt atcgccatgt 8400 aagttgttgg tccacggctc catttcgcac tgtcgtgact ggtcacgcga cgtatatcac 8460 aagcgccaaa tacgtcacga attccctttt ttaccgcata aggccaggag ccatcttcag 8520 ctggcgaatg ttcccaggcg cgatggaaag ccaaccatcc aagcaggcgt tcctgctcca 8580 tctgattgtt ttttaaatca ttaacgcgtt gttgttcagc tcggaggcgg cgtgcttcag 8640 cctggcgctc catgcgtgca cgttcttctt ccggctgagc gaccacggtc gcaccattcc 8700 gttgctgttc acggcgatac tccgaaaaca ggaatgaaaa gccactccag gagccagcgt 8760 catgcgcttt ttcaacgaag ttaacgaaag gataactgat gccatccttg ctctgctcaa 8820 ggcgtgaata gatttccaca cggcctttaa ggctcttctg cagagcttcc ggggaggaat 8880 tattgtaggt ggtatagcgc tctacaccac cgcgcggatt gagctgaatc ttatcagcac 8940 acgcaggcca gttgataccg gccatcttcg ccagctcagt cagctcatca cgtgccgcgt 9000 caagcagtga aaacggatcg ctgccaaagc gctccgcgta gaattcttgt aaggtcattt 9060 tttagccttt ccatgcgaat tagcattttt tcgggttgaa aaaatccgca ggagcagcca 9120 caataaacgc actatctttc tgaaggacgt atctgcgtta tcgtggctac ttcctgaaaa 9180 aggcccgagt ttgccgactc gggttttttt tcgtcttttt tcggctgcta cggtctggtt 9240 caaccccgac aaagtataga tcggattaaa ccagaattat agtcagcaat aaaccctgtt 9300 attgtatcat ctaccctcaa ccatgaacga tttgatcgta ccgactactt ggtgcacaaa 9360 ttgaagatca cttttatcat ggataacccg ttgagagtta gcactatcaa ggtagtaatg 9420 ctgctcgtca taacgggcta atcgttgaat tgtgatctcg ccgttattat cacaaaccag 9480 tacatcctca cccggtacaa gcgtaagtga agaatcgacc aggataacgt ctcccggctg 9540 gtagtttcgc tgaatctggt tcccgaccgt cagtgcgtaa acggtgttcc gttgactcac 9600 gaacggcagg aatcgctctg tgttggcagg ttctccaggc tgccagtctc tatccggtcc 9660 ggtctctgtc gtaccaataa caggaacgcg gtctggatca gattcagtgc catacagtat 9720 ccattgcacg ggcttacgca ggcattttgc cagcgatagc ccgatctcca gcgacggcat 9780 cacgtcgcca cgttctaagt tttggacgcc cggaagagag attcctacag cttctgccac 9840 ttgcttcagc gtcagtttca gctctaaacg gcgtgctttc agtcgttcgc ctcgtgtttt 9900 cataccctta atcataaatg atctctttat agctggctat aatttttata aattatacct 9960 agctttaatt ttcacttatt gattataata atccccatga aacccgaaga acttgtgcgc 10020 catttcggcg atgtggaaaa agcagcggtt ggcgtgggcg tgacacccgg cgcagtctat 10080 caatggctgc aagctgggga gattccacct ctacgacaaa gcgatataga ggtccgtacc 10140 gcgtacaaat taaagagtga tttcacctct cagcgcatgg gtaaggaagg gcataacagg 10200 ggatcctcta gagtcgacct gcaggcatgc aagcttcctg aatcgcccca tcatccagcc 10260 agaaagtgag ggagccacgg ttgatgagag ctttgttgta ggtggaccag ttggtgattt 10320 tgaacttttg ctttgccacg gaacggtctg cgttgtcggg aagatgcgtg atctgatcct 10380 tcaactcagc aaaagttcga tttattcaac aaagccgccg tcccgtcaag tcagcgtaat 10440 gctctgccag tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa 10500 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt 10560 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg 10620 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat 10680 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag 10740 cttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc 10800 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg 10860 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc 10920 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt 10980 tttccctggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt 11040 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac 11100 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc 11160 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc 11220 atataaatca gcatccatgt tggaatttaa tcgcggcctc gagcaagacg tttcccgttg 11280 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca 11340 tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct 11400 ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc ttcccgacaa 11460 cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca cctacaacaa 11520 agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga ttcaggaagc 11580 ttgcatgcct gcaggtcgac tctagaggat ccccgagaac ccgataatcg ctaccagtga 11640 tgatggctgt tttgcggcgg cgtgagccat cggcaatttc gataatgcct gacgtccttc 11700 tggcgaacgc ggggttctgc tgtcctgaag tgaggaatga agggataagg tcggccagcg 11760 ctgattcgtt cagcaattcc tgatcacgtt cattaccgag ccaaaccatt gtggcctttt 11820 cgactttatc agcaggaatg gtttccagct taaaagtcac gttgcggcca tcaagcttga 11880 attcgtacgc agaaaggccc acccgaaggt gagccagtgt gattacattt gcggccagtc 11940 cagttacgct ggagtcacta gtgcggccgc gacaacttgt ctagggccca atggcccggg 12000 aggcctactt aagtaagccg gcttagctag cgggacaggt ttcccgactg gaaagcgggc 12060 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac 12120 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gacaacaatt tcacacagga 12180 aacagctatg accatgatta cgccaagcta tttaggtgag actatagaat actcaagctt 12240 gcatgcgata cgtatcgtta acgatggatc cgacgcacgt gcgaattcgc cctatagtga 12300 gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 12360 cacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 12420 ggcccgcacc gatcgccctt cccaacagtt gcgcagctga atggcgaatg gcgcctgagc 12480 tagctggccc gggtggccca tgccagggcc attggcgcgc catgacttga agtcgcggcc 12540 gcactgacca tttaaatcat accaacatgg tcaaataaaa cgaaaggctc agtcgaaaga 12600 ctgggccttt cgttttaatc tgatcggcac gtaagaggtt ccaactttca ccataatgaa 12660 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct 12720 aaaatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 12780 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 12840 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagtttacgc 12900 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 12960 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 13020 ttggttgagt actcaccagt cacagaaaag catctcacgg atggcatgac agtaagagaa 13080 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctggcaacg 13140 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 13200 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 13260 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 13320 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg atcacttctg 13380 cgctcggccc tcccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 13440 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgcat cgtagttatc 13500 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 13560 gcctcactga ttaagcattg gtaatgacag aagtcaaaag cctccggtcg gaggcttttg 13620 actttctgct agatctgttt caatgcggtg aagggccagg cagctgggga ttatgtcgag 13680 acccggccag catgttggtt ttatcgcata ttcagcgttg tcgcgtttac ccaggtaaaa 13740 tggaagcagt gtatcgtctg cgtgaatgtg caaatcagga acgtaaccgt ggtacataga 13800 tgcagtccct tgcgggtcgt tcccttcaac gagtaggacg cggtgccctt gcaaggctaa 13860 ccattgcgcc tggtgtactg cagatgaggt tttataaacc cctcccttgt gtgacataac 13920 ggaaagtaca accgggtttt tatcgtcagg tctttggttt gggttaccaa acacactccg 13980 catatggcta atttggtcaa ttgtgtagcc agcgcgacgt tctactcggc ccctcatctc 14040 aaaatcagga gccggtagac gaccagcttt ttccgcgtct ctgatagcct gcggtgttac 14100 gccgatcagg tctgcaactt ctgttatacc ccagcggcga gtaatacgac gcgcttccgg 14160 gctgtcatcg ccgaactgtg cgatggcaat agcgcgcgtc atttcctgac cgcgattgat 14220 acagtctttc agcaaattaa ttaacgacat cctgtttcct ctcaaacatg cccttatctt 14280 tgtgtttttc atcatacttt acgtttttaa agcaaagcaa cataaaaaaa gcaaagtgac 14340 ttagaaaacg caaagttaag gttcaaatca attttttgat gcgctacaga agctatttag 14400 cttcatctaa gcgcaacggt attacttacg ttggtatatt taaaacctaa cttaatgatt 14460 ttaaatgata ataaatcata ccaattgcta tcaaaagtta agcgaacatg ctgattttca 14520 cgctgtttat acactttgag gcatctctat ctcttccgtc tctatattga aacacaatca 14580 aagaacatca atccatgtga catcccccac tatctaagaa caccataaca gaacacaaca 14640 taggaatgca acattaatgt atcaataatt cggaacatat gcactatatc atatctcaat 14700 tacggaacat atcagcacac aattgcccat tatacgc 14737 <210> 38 <211> 33 <212> DNA
<213> Artificial <220>
<223> lacFSfi <400> 38 cccaatggcc cgggaggcct acttaagtaa gcc 33 <210> 39 <211> 46 <212> DNA
<213> Artificial <220>
<223> lacRSfi <400> 39 tggcatgggc cacccgggcc agctagctca ggcgccattc gccatt 46 <210> 40 <211> 47 <212> DNA
<213> Artificial <220>
<223> PRD1 POL- F
<400> 40 aacagacagc agcatgccgc gccgttcccg taaaaaggtg gaatata 47 <210> 41 <211> 48 <212> DNA
<213> Artificial <220>
<223> PRD POL R

, <400> 41 ctgactctgg atatcttatt atgttccttt gattgtgcgc ttgataaa 48 <210> 42 <211> 28 <212> DNA
<213> Artificial <220>
<223> te1N-F
<400> 42 gcggatcccg atatccagag acttagaa 28 <210> 43 <211> 29 <212> DNA
<213> Artificial <220>
<223> te1N-R
<400> 43 cgaagcttct tttagctgta gtacgtttc 29 <210> 44 <211> 1833 <212> DNA
<213> Artificial <220>
<223> pSMART HCAmp <400> 44 gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga cctccatagc 60 agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa gaggttccaa 120 ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc 180 aggagctaag gaagctaaaa tgagtattca acatttccgt gtcgccctta ttcccttttt 240 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 300 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 360 ccttgagagt ttacgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 420 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 480 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc tcacggatgg 540 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 600 cttacttctg gcaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 660 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 720 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 780 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 840 tgcaggatca cttctgcgct cggccctccc ggctggctgg tttattgctg ataaatctgg 900 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 960 ccgcatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 1020 gatcgctgag ataggtgcct cactgattaa gcattggtaa tgagggccca aatgtaatca 1080 cctggctcac cttcgggtgg gcctttctgc gttgctggcg tttttccata ggctccgccc 1140 ccctgacgag catcacaaaa atcgatgctc aagtcagagg tggcgaaacc cgacaggact 1200 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 1260 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 1320 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 1380 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 1440 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1500 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1560 aagaacagta tttggtatct gcgctctgct gaagccagtt acctcggaaa aagagttggt 1620 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 1680 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgattttcta ccgaagaaag 1740 gcccacccgt gaaggtgagc cagtgagttg attgcagtcc agttacgctg gagtctgagg 1800 ctcgtcctga atgatatcaa gcttgaattc gtt 1833 <210> 45 <211> 14549 <212> DNA
<213> Artificial <220>
<223> pNZKC
<400> 45 gcgtataatg gactattgtg tgctgataag gagaacataa gcgcagaaca atatgtatct 60 attccggtgt tgtgttcctt tgttattctg ctattatgtt ctcttatagt gtgacgaaag 120 cagcataatt aatcgtcact tgttctttga ttgtgttacg atatccagag acttagaaac 180 gggggaaccg ggatgagcaa ggtaaaaatc ggtgagttga tcaacacgct tgtgaatgag 240 gtagaggcaa ttgatgcctc agaccgccca caaggcgaca aaacgaagag aattaaagcc 300 gcagccgcac ggtataagaa cgcgttattt aatgataaaa gaaagttccg tgggaaagga 360 ttgcagaaaa gaataaccgc gaatactttt aacgcctata tgagcagggc aagaaagcgg 420 tttgatgata aattacatca tagctttgat aaaaatatta ataaattatc ggaaaagtat 480 cctctttaca gcgaagaatt atcttcatgg ctttctatgc ctacggctaa tattcgccag 540 cacatgtcat cgttacaatc taaattgaaa gaaataatgc cgcttgccga agagttatca 600 aatgtaagaa taggctctaa aggcagtgat gcaaaaatag caagactaat aaaaaaatat 660 ccagattgga gttttgctct tagtgattta aacagtgatg attggaagga gcgccgtgac 720 tatctttata agttattcca acaaggctct gcgttgttag aagaactaca ccagctcaag 780 gtcaaccatg aggttctgta ccatctgcag ctaagccctg cggagcgtac atctatacag 840 caacgatggg ccgatgttct gcgcgagaag aagcgtaatg ttgtggttat tgactaccca 900 acatacatgc agtctatcta tgatattttg aataatcctg cgactttatt tagtttaaac 960 actcgttctg gaatggcacc tttggccttt gctctggctg cggtatcagg gcgaagaatg 1020 attgagataa tgtttcaggg tgaatttgcc gtttcaggaa agtatacggt taatttctca 1080 gggcaagcta aaaaacgctc tgaagataaa agcgtaacca gaacgattta tactttatgc 1140 gaagcaaaat tattcgttga attattaaca gaattgcgtt cttgctctgc tgcatctgat 1200 ttcgatgagg ttgttaaagg atatggaaag gatgatacaa ggtctgagaa cggcaggata 1260 aatgctattt tagcaaaagc atttaaccct tgggttaaat catttttcgg cgatgaccgt 1320 cgtgtttata aagatagccg cgctatttac gctcgcatcg cttatgagat gttcttccgc 1380 gtcgatccac ggtggaaaaa cgtcgacgag gatgtgttct tcatggagat tctcggacac 1440 gacgatgaga acacccagct gcactataag cagttcaagc tggccaactt ctccagaacc 1500 tggcgacctg aagttgggga tgaaaacacc aggctggtgg ctctgcagaa actggacgat 1560 gaaatgccag gctttgccag aggtgacgct ggcgtccgtc tccatgaaac cgttaagcag 1620 ctggtggagc aggacccatc agcaaaaata accaacagca ctctccgggc ctttaaattt 1680 agcccgacga tgattagccg gtacctggag tttgccgctg atgcattggg gcagttcgtt 1740 ggcgagaacg ggcagtggca gctgaagata gagacacctg caatcgtcct gcctgatgaa 1800 gaatccgttg agaccatcga cgaaccggat gatgagtccc aagacgacga gctggatgaa 1860 gatgaaattg agctcgacga gggtggcggc gatgaaccaa ccgaagagga agggccagaa 1920 gaacatcagc caactgctct aaaacccgtc ttcaagcctg caaaaaataa cggggacgga 1980 acgtacaaga tagagtttga atacgatgga aagcattatg cctggtccgg ccccgccgat 2040 agccctatgg ccgcaatgcg atccgcatgg gaaacgtact acagctaaaa gaaaagccac 2100 cggtgttaat cggtggcttt tttattgagg cctgtcccta cccatcccct gcaagggacg 2160 gaaggattag gcggaaactg cagctgcaac tacggacatc gccgtcccga ctgcagggac 2220 , ttccccgcgt aaagcggggc ttaaattcgg gctggccaac cctatttttc tgcaatcgct 2280 ggcgatgtta gtttcgtgga tagcgtttcc agcttttcaa tggccagctc aaaatgtgct 2340 ggcagcacct tctccagttc cgtatcaata tcggtgatcg gcagctctcc acaagacata 2400 ctccggcgac cgccacgaac tacatcgcgc agcagctccc gttcgtagac acgcatgttg 2460 cccagagccg tttctgcagc cgttaatatc cggcgcagct cggcgatgat tgccgggaga 2520 tcatccacgg ttattgggtt cggtgatggg ttcctgcagg cgcggcggag agccatccag 2580 acgccgctaa cccatgcgtt acggtactga aaactttgtg ctatgtcgtt tatcaggccc 2640 cgaagttctt ctttctgccg ccagtccagt ggttcaccgg cgttcttagg ctcaggctcg 2700 acaaaagcat actcgccgtt tttccggata gctggcagaa cctcgttcgt cacccacttg 2760 cggaaccgcc aggctgtcgt cccctgtttc accgcgtcgc ggcagcggag gattatggtg 2820 tagagaccag attccgatac cacatttact tccctggcca tccgatcaag tttttgtgcc 2880 tcggttaaac cgagggtcaa tttttcatca tgatccagct tacgcaatgc atcagaaggg 2940 ttggctatat tcaatgcagc acagatatcc agcgccacaa accacgggtc accaccgaca 3000 agaaccaccc gtatagggtg gctttcctga aatgaaaaga cggagagagc cttcattgcg 3060 cctccccgga tttcagctgc tcagaaaggg acagggagca gccgcgagct tcctgcgtga 3120 gttcgcgcgc gacctgcaga agttccgcag cttcctgcaa atacagcgtg gcctcataac 3180 tggagatagt gcggtgagca gagcccacaa gcgcttcaac ctgcagcagg cgttcctcaa 3240 tcgtctccag caggccctgg gcgtttaact gaatctggtt catgcgatca cctcgctgac 3300 cgggatacgg gctgacagaa cgaggacaaa acggctggcg aactggcgac gagcttctcg 3360 ctcggatgat gcaatggtgg aaaggcggtg gatatgggat tttttgtccg tgcggacgac 3420 agctgcaaat ttgaatttga acatggtatg cattcctatc ttgtataggg tgctaccacc 3480 agagttgaga atctctatag gggtggtagc ccagacaggg ttctcaacac cggtacaaga 3540 agaaaccggc ccaaccgaag ttggccccat ctgagccacc ataattcagg tatgcgcaga 3600 tttaacacac aaaaaaacac gctggcgcgt gttgtgcgct tcttgtcatt cggggttgag 3660 aggcccggct gcagattttg ctgcagcggg gtaactctac cgccaaagca gaacgcacgt 3720 caataattta ggtggatatt ttaccccgtg accagtcacg tgcacaggtg tttttatagt 3780 ttgctttact gactgatcag aacctgatca gttattggag tccggtaatc ttattgatga 3840 ccgcagccac cttagatgtt gtctcaaacc ccatacggcc acgaatgagc cactggaacg 3900 gaatagtcag caggtacagc ggaacgaacc acaaacggtt cagacgctgc cagaacgtcg 3960 catcacgacg ttccatccat tcggtattgt cgacgacctg gtaagcgtat tgtcctggcg 4020 tttttgctgc ttccgagtag caatcctctt caccacaaag aaagttactt atctgcttcc 4080 agttttcgaa cccttcttct ttgagccgct tttccagctc attcctccac aaaacaggca 4140 cccatcctct gcgataaatc atgattattt gtcctttaaa taaggctgta gaactgcaaa 4200 atcgctctcg ttcacatgct gtacgtagat gcgtagcaaa ttgccgttcc atccctgtaa 4260 tccaccttct ttggaaagat cgtccttgac ctcacgaaga accttatcca atagccctgc 4320 ggcacaagaa attgcctgct ctggatcagc aaattcatat tgattaatag gtgattgcca 4380 cacaccaaaa acaggaatca tcttttcggc taaacgcctc tcctgttctt tcttaatctc 4440 aagttgtaag cggaccagct caccatccat cattttttgt agatcatgcg ccactattca 4500 cccccactgg ccatcagcaa ataaagcttc atactcggac accggcaggc ggcttccacg 4560 gattgaaagg tcaagccaac cacgtccaga tgggtcagcc ttatccgatt cttcccaccg 4620 ttctgcagct gtagcaacca ggcattctac cgccttcatg tagtcttctg tacggaacca 4680 gccgtagtta atgccaccat cagtaactgc ccaggccatc tttttctctt cggcctcaat 4740 agcccggatg cggttatcgc acagctcgcg acagtacttc agctgttcgt aatccagttg 4800 cttcaggaac tctggtgtcg acgtcatagt ggcttcacct tataggcttt tagaagcgcc 4860 ctggcttcgt ctgtgtggtc ttccatgctc ttatcgctgg caatgcagca ataaactccc 4920 tcactatctg agaacccgtt catccgaatg atcgtgaatg gaagttcccg gccagtttta 4980 taatcgctat agcttgtcgc gtcgtggctg accttgacca cataagggtc gtagocctcc 5040 acgatgacaa ggcattcccg ttgttttccc attacccctc cggttatatc gccacggctt 5100 gccgctggct tagaaacgct ttcagcagcc ttatttcgcg tactgatagc aggtccataa 5160 attcggtcat gtacagcgag gcgaacgttc tcgcgatgct ggccactggc cacaggcgta 5220 ccgcctccat ttcggttgct ggcaacgcgt tctccgccca cgcctccggt accgccaccg 5280 ggatagcctc cagtgcctgg ataattactg attgtggggc gtccggaacg tgctctgttt 5340 tggatcgagg gttaccatgt atatctatat ttagatccaa atcgcgatcc acttcgatgg 5400 tggttttttc caccttacgt gcgtgaattg ataaaccggc ctcgcggcgc ttctccacga 5460 tattcatgag gaactcgacc gagtccgggt caatggaacg catcgtgggg cgtgcatcgc 5520 cgtctctggc gcgtctggtc ttactggata gccccataga ctccaggatg cctatgcaga 5580 ggtctgcagg cgctttcttc ttgcctttct ctgtgttgaa gccgccgatg cgtaaaacgt 5640 tgtttagcag atcgcgccgt tccggcgtga gcaggttatc tctggcgcgt ttgagggcgt 5700 ccatgtctgc ttcaccttcc agggtttttg gatcgatacc gcagtcgcgg aagtactgct 5760 gcagcgtcgc cgatttgagg gtgtagaaac cacgcatgcc tatctcaaca gcaggggtcg 5820 atttcactcg gtaatcggtt atggccggga atttagcctg gaactctgcg tcggcctgtt 5880 cccgcgtcat ggccgtagtg acgaactgct gccatcttcc ggcaacgcga taagcgtagg 5940 taaagtgaat caacgcttct tcacggtcaa ggcgacgggc ggttatctca tccagctgca 6000 tggtttcaaa caggcgcact tttttcaggc cgccgtcgaa atagaatttt aacgccacct 6060 cgtcgacatc cagctgcagc tccttttcga tgtcccagcg gaccagctgg gcctgctcat 6120 ccagggacag ggtgcgtttt tttatcaact catcgtgttc ggcctggtca ggagtatcga 6180 cactcaggtg gcgctccata agctgctcaa agaccagttc acgggcttct ttacgtaaat 6240 ccttaccgat gctgtttgca agcgcgtcgg tggccatagg cgcgacctga tagccatcat 6300 catgcatgat gcaaatcatg ttgctggcat aatcatttct ggccgatgcc tcgagcgcgg 6360 cggctttaat tttgagctgc atgaatgaag agttagccac gccgagtgaa attcggtcac 6420 cgtcaaagac aacgtctgtc agcagcccgg agtggccagc cgtttcgagc aaggcctgcg 6480 cgtaggcgcg tttgattttt tccggatcgg tttcacgttt accgcgaagc ttgtcgaaac 6540 cgataatgta ttcctgagct gtacggtcgc ggcgcagcat ctggatggcg tcgctgggga 6600 ccacttcgcc gcagaacatg ccgaaatggc ggtggaagtg tttctcctca atcgatacac 6660 ctgaagatat cgacgggctg tagatgaggc cgtcatattt tttcaccatc actttaggct 6720 ggttggtgaa atcgtcgact tccttctcct gtttgttttt ctggttaacg cagagaaact 6780 ttttgtcagg gaactgtagt ctcagctgca tggtaacgtc ttcggcgaac gtcgaactgt 6840 cggtggccag catgattcgt tcgccgcgtt gcactgcagc gataacctcg gtcatgatcc 6900 gatttttctc ggtataaaat acgcggatag gcttgttggt ttcgcggttg cgaacgtcga 6960 ccgggagttc aatcacgtga atttgcagcc aggcaggtag gcccagctcc tcgcgtcgct 7020 tcatcgccag ttcagccagg tcaacaagca gatcgttggc atcggcatcc accataatgg 7080 catgctcttc agtacgcgcc agcgcgtcga taagcgtgtt gaatacgcct accgggtttt 7140 ccatcgcacg cccggccaga atggcacgca ggccctgtgt tgcttcatcg aagccgaaga 7200 agtcatgctg gcgcatcagc ggttgccagc agcctttaag tatggagttg atgcaaatag 7260 tcagcttgtt ggcatatggc gccatttcct gatagccggg atcctgataa tgcagaatgt 7320 cggctttcgc gcctttccct tcggtcatca tttcatgcag gccgcctatc agggatacgc 7380 ggtgcgcgac ggaaacgcca cgcgtggact gcagcatcag tggacgcagg aggcctgtcg 7440 atttacccga ccccatcccg gcgcggacaa taacgatgcc ctgcagctgt gcggcgtatg 7500 tcatcacctc atcggtcatc ctggaggttt caaaccgttt gtaagtgatg tgtgacgggc 7560 gaaggttcgg gttggtgatg cgttcactga acgaacgtga tgtttgcgcg gcacggcatt 7620 tgcgattcaa ccggcgcgta atgtgatctt taacggtacc gttataaatt tctgcgatac 7680 ccatatcccg cagcgtgctg cttaaaaggc gcataagttc tttcgggctg tttggtaccg 7740 ggcatgtcag catgccaata tcaacggcgc gaagcagttc tttggcaaaa gtgcgtctgt 7800 tcagacgcgg gagagtacgc agcttattca gcgtgatcga caacagatcg gttgcacggc 7860 tcagatgatt tctcgttaat ctggcgagcg acttccttca gccctctcag gctgtgcagg 7920 tcgttaaaat cgctgcattc cagctcaggg tcatcctcaa aagttgggta aacacatttg 7980 acgccggaaa acttctccat gatgtcgaat ccggtgcgga ggcctgtgtt gccttttcct 8040 tcagctgagg atttgcggtc gttatcgaga gcgcaagtga tttgcgcagc cgggtacatg 8100 ttcaccagct gctcgacaac gtgaatcatg ttgttagcgg aaaccgcaat gactaccgcg 8160 tcaaagcgtt ttttcgggtc gtttctggtc gccagccaga tggatgcccc ggtggcgaaa 8220 ccctctgcag tcgcaatttt ttgcgccccc tgcaggtcgc caataacaaa gcatgcaccg 8280 acgaaatcac cgttagtgat ggcgctggtc tggaacttgc caccattcag atcgatacgt 8340 tgccagccaa caatccgccc gtcttttctt ccgtccaggt gggacagagg tatcgccatg 8400 taagttgttg gtccacggct ccatttcgca ctgtcgtgac tggtcacgcg acgtatatca 8460 caagcgccaa atacgtcacg aattcccttt tttaccgcat aaggccagga gccatcttca 8520 gctggcgaat gttcccaggc gcgatggaaa gccaaccatc caagcaggcg ttcctgctcc 8580 atctgattgt tttttaaatc attaacgcgt tgttgttcag ctcggaggcg gcgtgcttca 8640 gcctggcgct ccatgcgtgc acgttcttct tccggctgag cgaccacggt cgcaccattc 8700 cgttgctgtt cacggcgata ctccgaaaac aggaatgaaa agccactcca ggagccagcg 8760 tcatgcgctt tttcaacgaa gttaacgaaa ggataactga tgccatcctt gctctgctca 8820 aggcgtgaat agatttccac acggccttta aggctcttct gcagagcttc cggggaggaa 8880 ttattgtagg tggtatagcg ctctacacca ccgcgcggat tgagctgaat cttatcagca 8940 cacgcaggcc agttgatacc ggccatcttc gccagctcag tcagctcatc acgtgccgcg 9000 tcaagcagtg aaaacggatc gctgccaaag cgctccgcgt agaattcttg taaggtcatt 9060 ttttagcctt tccatgcgaa ttagcatttt ttcgggttga aaaaatccgc aggagcagcc 9120 acaataaacg cactatcttt ctgaaggacg tatctgcgtt atcgtggcta cttcctgaaa 9180 aaggcccgag tttgccgact cgggtttttt ttcgtctttt ttcggctgct acggtctggt 9240 tcaaccccga caaagtatag atcggattaa accagaatta tagtcagcaa taaaccctgt 9300 tattgtatca tctaccctca accatgaacg atttgatcgt accgactact tggtgcacaa 9360 attgaagatc acttttatca tggataaccc gttgagagtt agcactatca aggtagtaat 9420 gctgctcgtc ataacgggct aatcgttgaa ttgtgatctc gccgttatta tcacaaacca 9480 gtacatcctc acccggtaca agcgtaagtg aagaatcgac caggataacg tctcccggct 9540 , ggtagtttcg ctgaatctgg ttcccgaccg tcagtgcgta aacggtgttc cgttgactca 9600 cgaacggcag gaatcgctct gtgttggcag gttctccagg ctgccagtct ctatccggtc 9660 cggtctctgt cgtaccaata acaggaacgc ggtctggatc agattcagtg ccatacagta 9720 tccattgcac gggcttacgc aggcattttg ccagcgatag cccgatctcc agcgacggca 9780 tcacgtcgcc acgttctaag ttttggacgc ccggaagaga gattcctaca gcttctgcca 9840 cttgcttcag cgtcagtttc agctctaaac ggcgtgcttt cagtcgttcg cctcgtgttt 9900 tcataccctt aatcataaat gatctcttta tagctggcta taatttttat aaattatacc 9960 tagctttaat tttcacttat tgattataat aatccccatg aaacccgaag aacttgtgcg 10020 ccatttcggc gatgtggaaa aagcagcggt tggcgtgggc gtgacacccg gcgcagtcta 10080 tcaatggctg caagctgggg agattccacc tctacgacaa agcgatatag aggtccgtac 10140 cgcgtacaaa ttaaagagtg atttcacctc tcagcgcatg ggtaaggaag ggcataacag 10200 gggatcctct agagtcgacc tgcaggcatg caagcttcct gaatcgcccc atcatccagc 10260 cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca gttggtgatt 10320 ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc 10380 ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa 10440 tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca 10500 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt 10560 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc 10620 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa 10680 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa 10740 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat 10800 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc 10860 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg 10920 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg 10980 ttttccctgg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct 11040 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa 11100 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc 11160 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc 11220 catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt 11280 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc 11340 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc 11400 tttgttgaat aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttoccgaca 11460 acgcagaccg ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca 11520 aagctctcat caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggaag 11580 cttgcatgcc tgcaggtcga ctctagagga tccccgagaa cccgataatc gctaccagtg 11640 atgatggctg ttttgcggcg gcgtgagcca tcggcaattt cgataatgcc tgacgtcctt 11700 ctggcgaacg cggggttctg ctgtcctgaa gtgaggaatg aagggataag gtcggccagc 11760 gctgattcgt tcagcaattc ctgatcacgt tcattaccga gccaaaccat tgtggccttt 11820 tcgactttat cagcaggaat ggtttccagc ttaaaagtca cgttgcggcc atcaagcttg 11880 aattcgtacg cagaaaggcc cacccgaagg tgagccagtg tgattacatt tgcggccagt 11940 ccagttacgc tggagtcact agtgcggccg cgacaacttg tctagggccc aatggcccgg 12000 gaggcctact taagtaagcc ggcttagcta gcgggacagg tttcccgact ggaaagcggg 12060 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 12120 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggacaacaat ttcacacagg 12180 aaacagctat gaccatgatt acgccaagct atttaggtga gactatagaa tactcaagct 12240 tgcatgcgat acgtatcgtt aacgatggat ccgacgcacg tgcgaattcg ccctatagtg 12300 agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 12360 tcacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 12420 aggcccgcac cgatcgccct tcccaacagt tgcgcagctg aatggcgaat ggcgcctgag 12480 ctagcattgc cggcattctt aagtaggcct cccgggccat tggcgcgcca gacttgaagt 12540 cgcggccgca ctgaccattt aaatcatacc aacatggtca aataaaacga aaggctcagt 12600 cgaaagactg ggcctttcgt tttaatctga tcggcacgta agaggttcca actttcacca 12660 taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa 12720 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca 12780 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt 12840 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc 12900 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc gtatggcaat 12960 gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga 13020 gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct 13080 acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg 13140 gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga 13200 tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta 13260 tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga 13320 tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg 13380 cggggcgtaa cctaggtgac agaagtcaaa agcctccggt cggaggcttt tgactttctg 13440 ctagatctgt ttcaatgcgg tgaagggcca ggcagctggg gattatgtcg agacccggcc 13500 agcatgttgg ttttatcgca tattcagcgt tgtcgcgttt acccaggtaa aatggaagca 13560 gtgtatcgtc tgcgtgaatg tgcaaatcag gaacgtaacc gtggtacata gatgcagtcc 13620 cttgcgggtc gttcccttca acgagtagga cgcggtgccc ttgcaaggct aaccattgcg 13680 cctggtgtac tgcagatgag gttttataaa cccctccctt gtgtgacata acggaaagta 13740 caaccgggtt tttatcgtca ggtctttggt ttgggttacc aaacacactc cgcatatggc 13800 taatttggtc aattgtgtag ccagcgcgac gttctactcg gcccctcatc tcaaaatcag 13860 gagccggtag acgaccagct ttttccgcgt ctctgatagc ctgcggtgtt acgccgatca 13920 ggtctgcaac ttctgttata ccccagcggc gagtaatacg acgcgcttcc gggctgtcat 13980 cgccgaactg tgcgatggca atagcgcgcg tcatttcctg accgcgattg atacagtctt 14040 tcagcaaatt aattaacgac atcctgtttc ctctcaaaca tgcccttatc tttgtgtttt 14100 tcatcatact ttacgttttt aaagcaaagc aacataaaaa aagcaaagtg acttagaaaa 14160 cgcaaagtta aggttcaaat caattttttg atgcgctaca gaagctattt agcttcatct 14220 aagcgcaacg gtattactta cgttggtata tttaaaacct aacttaatga ttttaaatga 14280 taataaatca taccaattgc tatcaaaagt taagcgaaca tgctgatttt cacgctgttt 14340 atacactttg aggcatctct atctcttccg tctctatatt gaaacacaat caaagaacat 14400 caatccatgt gacatccccc actatctaag aacaccataa cagaacacaa cataggaatg 14460 caacattaat gtatcaataa ttcggaacat atgcactata tcatatctca attacggaac 14520 atatcagcac acaattgccc attatacgc 14549

Claims (20)

1. A linear cloning vector replicable in a bacterial host, comprising:
a) a left arm comprising a left telomere and a first selectable marker;
b) a right arm comprising a right telomere and a second selectable marker;
and c) a cloning region located between the left arm and the right arm.

2. The vector of claim 1, further comprising a pair of transcriptional terminator regions flanking the cloning region, wherein the transcriptional terminator regions are the same or different and are unidirectional or bidirectional.

3. The vector of claim 2, further comprising a transcriptional terminator region after the second selectable marker.

4. The vector of claim 1, wherein the cloning region comprises a multiple cloning region.

5. The vector of claim 1, wherein the cloning region comprises a stuffer region.

6. The vector of claim 5, wherein the stuffer region comprises a reporter gene flanked by a pair of restriction sites which are the same or different.

7. The vector of any one of claims 1-6, further comprising one or more vector arm genes, each vector arm gene being in either the left arm or the right arm, wherein the one or more vector arm genes comprises a gene encoding a product selected from the group consisting of a replication factor, a protelomerase, and a copy number regulator.

8. The vector of claim 7, wherein the one or more vector arm genes comprises at least a gene encoding a replication factor and a gene encoding a copy number regulator.

9. The vector of claim 7 or claim 8, wherein the replication factor is repA
and the copy number regulator is cB.

10. The vector of any one of claims 1-9, wherein the cloning region comprises an element selected from the group consisting of a unique restriction site and a pair of identical restriction sites, and wherein the element is not included within the arms.

11. The vector of any one of claims 1-10, wherein the vector is replicable in E. coli.

12. A host cell comprising the vector of any one of claims 1-11.

13. The host cell of claim 12, further comprising a polynucleotide sequence encoding a protelomerase.

14. The host cell of claim 13, wherein the polynucleotide sequence encoding the protelomerase is integrated into the host cell genome.

15. The host cell of claim 13, further comprising a polynucleotide sequence encoding a partitioning protein, a polynucleotide sequence encoding an antirepressor, or combinations thereof.

16. A kit comprising the linear cloning vector of any one of claims 1-11 and a recombinant host cell comprising a polynucleotide sequence encoding a protelomerase, wherein the polynucleotide sequence is integrated into the host cell genome.

17. A method of cloning a polynucleotide sequence having a first end and a second end comprising:
a) processing the linear cloning vector of any one of claims 1-11 to separate the right arm from the left arm;
b) joining the first end of the polynucleotide sequence to the right arm and the second end of the polynucleotide sequence to the left arm to provide a joined product;
c) transforming a host cell with the ligation product; and d) growing the transformed host cell on medium, such that selection is provided for the first and second selectable markers of the linear cloning vector.

18. The method of claim 17, wherein the host cell used in step c) is a recombinant host cell comprising a polynucleotide sequence encoding a protelomerase, wherein the polynucleotide sequence is integrated into the host cell genome.

19. A method of cloning at least two distinct polynucleotides comprising:
a) processing each of the polynucleotides to provide a linking sequence on both termini of the polynucleotides;
b) processing the linear cloning vector of any one of claims 1-11 to separate the right arm from the left arm and to provide a linking sequence on the terminus opposite the telomere of each arm;
c) forming a joined product comprising the polynucleotides and the right and left arms, wherein the arms are noncontiguous with each other and are separated by the polynucleotides;
d) transforming a host cell with the joined product; and e) growing the transformed host cell on medium, such that selection is provided for the first and second selectable markers of the linear cloning vector, wherein multiplication of the host cell results in cloning of the polynucleotides.

20. The method of claim 19, further comprising restricting the cloned polynucleotides to verify presence and orientation of the cloned polynucleotides in the vector.